Muscle targeting complexes and uses thereof for treating dystrophinopathies

ABSTRACT

Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide, e.g., an oligonucleotide that causes exon skipping in a mRNA expressed from a mutant DMD allele.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 63/220,262, filed Jul. 9, 2021,entitled “MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATINGDYSTROPHINOPATHIES,” which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present application relates to targeting complexes for deliveringmolecular payloads (e.g., oligonucleotides) to cells and uses thereof,particularly uses relating to treatment of disease.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(D082470070US01-SEQ-ZJG.xml; Size: 2,660,754 bytes; and Date ofCreation: Jul. 1, 2022) is herein incorporated by reference in itsentirety.

BACKGROUND OF INVENTION

Dystrophinopathies are a group of distinct neuromuscular diseases thatresult from mutations in dystrophin gene. Dystrophinopathies includeDuchenne muscular dystrophy, Becker muscular dystrophy, and X-linkeddilated cardiomyopathy. Dystrophin (DMD) is a large gene, containing 79exons and about 2.6 million total base pairs. Numerous mutations in DMD,including exonic frameshift, deletion, substitution, and duplicativemutations, are able to diminish the expression of functional dystrophin,leading to dystrophinopathies. One agent that targets exon 51 of humanDMD, eteplirsen, has been preliminarily approved by the U.S. Food andDrug Administration (FDA) however its efficacy is still being evaluated.

SUMMARY OF INVENTION

According to some aspects, the disclosure provides complexes that targetmuscle cells for purposes of delivering molecular payloads to thosecells. In some embodiments, complexes provided herein are particularlyuseful for delivering molecular payloads that increase or restoreexpression or activity of functional DMD. In some embodiments, complexescomprise oligonucleotide based molecular payloads that promote normalexpression of functional DMD through an in-frame exon skipping mechanismor suppression of stop codons. In other embodiments, complexes areconfigured for delivering a mini-dystrophin gene or synthetic mRNA thatincreases or restores functional dystrophin activity. Accordingly, insome embodiments, complexes provided herein comprise muscle-targetingagents (e.g., muscle targeting antibodies) that specifically bind toreceptors on the surface of muscle cells for purposes of deliveringmolecular payloads to the muscle cells. In some embodiments, thecomplexes are taken up into the cells via a receptor mediatedinternalization, following which the molecular payload may be releasedto perform a function inside the cells. For example, complexesengineered to deliver oligonucleotides may release the oligonucleotidessuch that the oligonucleotides can promote expression of functional DMD(e.g., through an exon skipping mechanism) in the muscle cells. In someembodiments, the oligonucleotides are released by endosomal cleavage ofcovalent linkers connecting oligonucleotides and muscle-targeting agentsof the complexes.

One aspect of the present disclosure relates to a complex comprising ananti-transferrin receptor (TfR) antibody covalently linked to amolecular payload configured for promoting the expression or activity ofa DMD gene, wherein the antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 76; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 75;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 69; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 74;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 75;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 74;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 80.

In some embodiments, the antibody comprises:

(i) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(ii) a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(iii) a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(iv) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(v) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VLcomprising the amino acid sequence of SEQ ID NO: 74;

(vi) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(vii) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 74;

(viii) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VLcomprising the amino acid sequence of SEQ ID NO: 78;

(ix) a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VLcomprising the amino acid sequence of SEQ ID NO: 80; or

(x) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the antibody is selected from the group consistingof a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv, a Fv,and a full-length IgG. In some embodiments, the antibody is a Fabfragment.

In some embodiments, the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 97; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 98; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 99; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 103; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

In some embodiments, the antibody comprises:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101;and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO:101; and a light chain comprising the amino acid sequence of SEQ ID NO:89;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO:102; and a light chain comprising the amino acid sequence of SEQ ID NO:93;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103;and a light chain comprising the amino acid sequence of SEQ ID NO: 95;or

(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102;and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the antibody does not specifically bind to thetransferrin binding site of the transferrin receptor and/or themuscle-targeting antibody does not inhibit binding of transferrin to thetransferrin receptor.

In some embodiments, the molecular payload is an oligonucleotide. Insome embodiments, the oligonucleotide promotes exon skipping in a DMDRNA. In some embodiments, the oligonucleotide promotes skipping of anexon of DMD in the range of exon 8 to exon 55. In some embodiments, theoligonucleotide promotes skipping of exon 8, exon 23, exon 43, exon 44,exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and/or exon 55.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to one or more full or partial exonic splicing enhancers(ESE) of a DMD transcript. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprisingone or more full or partial ESEs as set forth in SEQ ID NOs: 402-436 and2043-2238.

In some embodiments, the oligonucleotide promotes skipping of exon 51.

In some embodiments, the oligonucleotide is 20-30 nucleotides in lengthand comprises a region of complementarity to a target sequencecomprising at least 4 consecutive nucleotides of an ESE as set forth inany one of SEQ ID NOs: 402-436.

In some embodiments, the oligonucleotide comprises any one of SEQ IDNOs: 437-1241, or comprises a region of complementarity to any one ofSEQ ID NOs: 1242-2046.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence of an oligonucleotide listed inTable 14. In some embodiments, the oligonucleotide comprises a sequencelisted in Table 14, wherein any one or more of the uracil bases (U's) inthe oligonucleotide may optionally be a thymine base (T).

In some embodiments, the oligonucleotide comprises at least one modifiedinternucleoside linkage. In some embodiments, the at least one modifiedinternucleoside linkage is a phosphorothioate linkage.

In some embodiments, the oligonucleotide comprises one or more modifiednucleosides. In some embodiments, the one or more modified nucleosidesare 2′-modified nucleosides.

In some embodiments, the oligonucleotide comprises one or morephosphorodiamidate morpholinos, optionally wherein the oligonucleotideis a phosphorodiamidate morpholino oligomer (PMO).

In some embodiments, the antibody is covalently linked to the molecularpayload via a cleavable linker. In some embodiments, the cleavablelinker comprises a valine-citrulline sequence.

In some embodiments, the antibody is covalently linked to the molecularpayload via conjugation to a lysine residue or a cysteine residue of theantibody.

Another aspect of the present disclosure relates to a method ofpromoting the expression or activity of a DMD protein in a cell, themethod comprising contacting the cell with a complex disclosed herein inan amount effective for promoting internalization of the molecularpayload to the cell, optionally wherein the cell is a muscle cell.

Another aspect of the present disclosure relates to a method of treatinga subject having a mutated DMD allele that is associated with adystrophinopathy, the method comprising administering to the subject aneffective amount of a complex disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a non-limiting schematic showing the effect oftransfecting cells with an siRNA.

FIG. 2 depicts a non-limiting schematic showing the activity of a muscletargeting complex comprising an siRNA.

FIGS. 3A-3B depict non-limiting schematics showing the activity of amuscle targeting complex comprising an siRNA in mouse muscle tissues(gastrocnemius and heart) in vivo, relative to control non-targetingcomplex comprising the same siRNA. (N=4 C57BL/6 WT mice)

FIGS. 4A-4E depict non-limiting schematics showing the tissueselectivity of a muscle targeting complex comprising an siRNA.

FIG. 5 depicts a non-limiting schematic showing the ability of ananti-transferrin receptor muscle targeting complex comprising an exon-23skipping phosphorodiamidate morpholino oligomer (PMO) todose-dependently enhance exon skipping in muscle tissues of a mdx mousemodel.

FIGS. 6A-6B depict non-limiting schematics showing the ability of ananti-transferrin receptor muscle targeting complex comprising an exon-23skipping PMO to dose-dependently increase dystrophin in skeletal muscle(quadriceps) of a mdx mouse model.

FIGS. 7A-7E depict non-limiting schematics showing the ability of ananti-transferrin receptor muscle targeting complex comprising an exon-23skipping PMO to improve functional performance (FIGS. 7A, 7B, 7C, and7D) and reduce creatine kinase levels (FIG. 7E) in an mdx mouse model.(**p<0.01; ***p<0.001; ****; p<0.0001; NS not significant)

FIG. 8 shows the serum stability of the linker used for linking ananti-TfR antibody and a molecular payload (e.g., an oligonucleotide) invarious species over time after intravenous administration.

FIGS. 9A-9F show binding of humanized anti-TfR Fabs to human TfR1(hTfR1) or cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. FIG. 9Ashows binding of humanized 3M12 variants to hTfR1. FIG. 9B shows bindingof humanized 3M12 variants to cTfR1. FIG. 9C shows binding of humanized3A4 variants to hTfR1. FIG. 9D shows binding of humanized 3A4 variantsto cTfR1. FIG. 9E shows binding of humanized 5H12 variants to hTfR1.FIG. 9F shows binding of humanized 5H12 variants to hTfR1.

FIG. 10 shows the quantified cellular uptake of anti-TfR Fab conjugatesinto rhabdomyosarcoma (RD) cells. The molecular payload in the testedconjugates are DMPK-targeting oligonucleotides and the uptake of theconjugates were facilitated by indicated anti-TfR Fabs. Conjugateshaving a negative control Fab (anti-mouse TfR) or a positive control Fab(anti-human TfR1) are also included this assay. Cells were incubatedwith indicated conjugate at a concentration of 100 nM for 4 hours.Cellular uptake was measured by mean Cypher5e fluorescence.

FIGS. 11A-11F show binding of oligonucleotide-conjugated or unconjugatedhumanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1(cTfR1), as measured by ELISA. FIG. 11A shows the binding of humanized3M12 variants alone or in conjugates with a DMPK targeting oligo tohTfR1. FIG. 11B shows the binding of humanized 3M12 variants alone or inconjugates with a DMPK targeting oligo to cTfR1. FIG. 11C shows thebinding of humanized 3A4 variants alone or in conjugates with a DMPKtargeting oligo to hTfR1. FIG. 11D shows the binding of humanized 3A4variants alone or in conjugates with a DMPK targeting oligo to cTfR1.FIG. 11E shows the binding of humanized 5H12 variants alone or inconjugates with a DMPK targeting oligo to hTfR1. FIG. 11F shows thebinding of humanized 5H12 variants alone or in conjugates with a DMPKtargeting oligo to cTfR1. The respective EC₅₀ values are also shown.

FIG. 12 shows DMPK expression in RD cells treated with variousconcentrations of conjugates containing the indicated humanized anti-TfRantibodies conjugated to a DMPK-targeting oligonucleotide ASO300. Theduration of treatment was 3 days. ASO300 delivered using transfectionagents were used as control.

FIG. 13 shows skipping of exon 51 in human DMD myotubes, facilitated bya DMD exon 51 skipping oligonucleotide (a PMO). Cells were treated withthe naked PMO or with PMO conjugated to an anti-TfR1 Fab (Ab-PMO).

FIG. 14 shows dose-dependent increase of dystrophin expression inquadriceps muscles of mdx mice after treatment with anti-mouse TfR1 (RI7217) conjugated to an oligonucleotide (a PMO) targeted to exon 23, asmeasured by western blotting for dystrophin, with alpha-actin as aloading control. The standards were generated using pooled wild-typeprotein and pooled mdx protein. The percent indicates the amount of WTprotein spiked into the sample.

FIG. 15 shows quantification of dystrophin protein levels withinquadriceps muscles of mdx mice after treatment with various doses ofanti-mouse TfR (RI7 217) conjugated to an oligonucleotide (a PMO)targeting exon 23.

FIG. 16 shows immunofluorescent staining images of quadriceps musclesfrom wild-type (WT) mice treated with saline, or mdx mice treated withsaline, naked oligonucleotide or oligonucleotide conjugated toanti-mouse TfR1 (RI7 217).

FIG. 17 shows data illustrating that conjugates containing designatedanti-TfR Fabs (3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3 N54S/VK4)conjugated to a DMD exon-skipping oligonucleotide resulted in enhancedexon skipping compared to the naked DMD exon skipping oligo in DMDpatient myotubes.

FIG. 18 shows ELISA measurements of binding of anti-TfR Fab 3M12 VH4/Vk3to recombinant human (circles), cynomolgus monkey (squares), mouse(upward triangles), or rat (downward triangles) TfR1 protein, at a rangeof concentrations from 230 pM to 500 nM of the Fab. Measurement resultsshow that the anti-TfR Fab is reactive with human and cynomolgus monkeyTfR1. Binding was not observed to mouse or rat recombinant TfR1. Data isshown as relative fluorescent units normalized to baseline.

FIG. 19 shows results of an ELISA testing the affinity of anti-TfR Fab3M12 VH4/Vk3 to recombinant human TfR1 or TfR2 over a range ofconcentrations from 230 pM to 500 nM of Fab. The data are presented asrelative fluorescence units normalized to baseline. The resultsdemonstrate that the Fab does not bind recombinant human TfR2.

FIG. 20 shows the serum stability of the linker used for linkinganti-TfR Fab 3M12 VH4/Vk3 to a control antisense oligonucleotide over 72hours incubation in PBS or in rat, mouse, cynomolgus monkey or humanserum.

FIGS. 21A-21C show quantification of exon 23 skipping in quadriceps(FIG. 21A), heart (FIG. 21B), and diaphragm (FIG. 21C) of wild-type (WT)and mdx mice two- or four-weeks following administration of a singledose of saline, unconjugated oligonucleotide (ASO) that induces exon 23skipping in DMD, or conjugates containing an anti-TfR RI7217 Fabconjugated to the ASO (Ab-ASO). Little or no exon 23 skipping wasobserved in tissues from WT mice or from mdx mice administered saline orunconjugated ASO, whereas significant levels of exon 23 skipping wasobserved in tissues of mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01,****p<0.0001)

FIGS. 22A-22D show measurement of dystrophin protein in quadriceps ofmdx mice following administration of a single dose of unconjugatedoligonucleotide (ASO) that induces exon 23 skipping in DMD, orconjugates containing an anti-TfR1 RI7217 Fab conjugated to the ASO(Ab-ASO). FIG. 22A shows western blots of dystrophin and alpha-actininprotein in muscle tissue two weeks following injection of ASO or Ab-ASO.FIG. 22B shows quantification of the dystrophin in the western blot ofFIG. 23A relative to dystrophin protein in wild-type muscle. FIG. 22Cshows western blots of dystrophin and alpha-actinin protein in muscletissue four weeks following injection of ASO or Ab-ASO. FIG. 22D showsquantification of the dystrophin in the western blot of FIG. 22Crelative to dystrophin protein in wild-type muscle. The standard curvesin FIGS. 22A and 22C were generated by pooling tissue from wild-type(WT) and mdx mouse samples, and the percent WT indicates the amount ofWT protein spiked into each sample. (*p<0.05; ns, not significant)

FIGS. 23A-23D show measurement of dystrophin protein in heart muscle ofmdx mice following administration of a single dose of unconjugatedoligonucleotide (ASO) that induces exon 23 skipping in DMD, orconjugates containing an anti-TfR1 RI7217 Fab conjugated to the ASO(Ab-ASO). FIG. 23A shows western blots of dystrophin and alpha-actininprotein in muscle tissue two weeks following injection of ASO or Ab-ASO.FIG. 23B shows quantification of the dystrophin in the western blot ofFIG. 23A relative to dystrophin protein in wild-type muscle. FIG. 23Cshows western blots of dystrophin and alpha-actinin protein in muscletissue four weeks following injection of ASO or Ab-ASO. FIG. 23D showsquantification of the dystrophin in the Western blot of FIG. 23Crelative to dystrophin protein in wild-type muscle. The standard curvesin FIGS. 23A and 23C were generated by pooling tissue from wild-type(WT) and mdx mouse samples, and the percent WT indicates the amount ofWT protein spiked into each sample. (*p<0.05, ****p<0.0001)

FIGS. 24A-24D show measurement of dystrophin protein in diaphragm muscleof mdx mice following administration of a single dose of unconjugatedoligonucleotide (ASO) that induces exon 23 skipping in DMD, orconjugates containing an anti-TfR1 RI7217 Fab conjugated to the ASO(Ab-ASO). FIG. 24A shows western blots of dystrophin and alpha-actininprotein in muscle tissue two weeks following injection of ASO or Ab-ASO.FIG. 24B shows quantification of the dystrophin in the western blot ofFIG. 24A relative to dystrophin protein in wild-type muscle. FIG. 24Cshows western blots of dystrophin and alpha-actinin protein in muscletissue four weeks following injection of ASO or Ab-ASO. FIG. 24D showsquantification of the dystrophin in the Western blot of FIG. 24Crelative to dystrophin protein in wild-type muscle. The standard curvesin FIGS. 24A and 24C were generated by pooling tissue from wild-type(WT) and mdx mouse samples, and the percent WT indicates the amount ofWT protein spiked into each sample. (**p<0.01, ***p<0.001)

FIGS. 25A-25C show quantification of the amount of administeredoligonucleotide (ASO) in quadriceps (FIG. 25A), diaphragm (FIG. 25B),and heart (FIG. 25C) of wild-type (WT) or mdx mice two- or four-weeksfollowing administration of a single dose of saline, unconjugated exon23 skipping oligonucleotide (ASO), or conjugates containing an anti-TfR1RI7217 Fab conjugated to the ASO (Ab-ASO).

FIG. 26 shows % exon 53 skipping in DMD patient cells harboring adeletion of DMD exon 52, following gymnotic uptake of exon 53-skippingoligonucleotides over a range of concentrations.

FIG. 27 shows % exon 53 skipping in DMD patient cells harboring adeletion of DMD exon 52, following treatment with exon 53-skipping PMOeither not linked to an antibody (“Naked ASO”) or covalently linked toan anti-TfR1 Fab (“Anti-TfR1 Fab-ASO complex”) at a variety ofconcentrations.

DETAILED DESCRIPTION OF INVENTION

Aspects of the disclosure relate to a recognition that while certainmolecular payloads (e.g., oligonucleotides, peptides, small molecules)can have beneficial effects in muscle cells, it has proven challengingto effectively target such cells. As described herein, the presentdisclosure provides complexes comprising muscle-targeting agentscovalently linked to molecular payloads in order to overcome suchchallenges. In some embodiments, the complexes are particularly usefulfor delivering molecular payloads that modulate (e.g., promote) theexpression or activity of target genes in muscle cells, e.g., in asubject having or suspected of having a rare muscle disease. Forexample, in some embodiments, complexes are provided for targeting DMD,e.g., a mutated DMD allele. In some embodiments, complexes providedherein may comprise oligonucleotides that promote normal expression andactivity of DMD. As another example, complexes may compriseoligonucleotides that induce skipping of exon of DMD mRNA. In someembodiments, synthetic nucleic acid payloads (e.g., DNA or RNA payloads)may be used that express one or more proteins that promote normalexpression and activity of DMD.

In some embodiments, complexes may comprise molecular payloads ofsynthetic cDNAs and/or (e.g., and) synthetic mRNAs, e.g., that expressdystrophin or fragments thereof (e.g., a dystrophin mini gene). In someembodiments, complexes may comprise molecular payloads such as guidemolecules (e.g., guide RNAs) that are capable of targeting nucleic acidprogrammable nucleases (e.g., Cas9) to a sequence at or near adisease-associated mutation of DMD, e.g., a mutated DMD exon. In someembodiments, such nucleic programmable nucleases could be used to cleavepart or all of a disease-associated mutation of DMD, e.g., a mutated DMDexon, to promote expression of functional DMD. In some embodiments,complexes may comprise molecular payloads that upregulate the expressionand/or (e.g., and) activity of genes that can replace the function ofdystrophin, such as utrophin.

Further aspects of the disclosure, including a description of definedterms, are provided below.

I. Definitions

Administering: As used herein, the terms “administering” or“administration” means to provide a complex to a subject in a mannerthat is physiologically and/or (e.g., and) pharmacologically useful(e.g., to treat a condition in the subject).

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless in either direction (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes at least one immunoglobulin variable domain or at leastone antigenic determinant, e.g., paratope that specifically binds to anantigen. In some embodiments, an antibody is a full-length antibody. Insome embodiments, an antibody is a chimeric antibody. In someembodiments, an antibody is a humanized antibody. However, in someembodiments, an antibody is a Fab fragment, a Fab′ fragment, a F(ab′)2fragment, a Fv fragment or a scFv fragment. In some embodiments, anantibody is a nanobody derived from a camelid antibody or a nanobodyderived from shark antibody. In some embodiments, an antibody is adiabody. In some embodiments, an antibody comprises a framework having ahuman germline sequence. In another embodiment, an antibody comprises aheavy chain constant domain selected from the group consisting of IgG,IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, andIgE constant domains. In some embodiments, an antibody comprises a heavy(H) chain variable region (abbreviated herein as VH), and/or (e.g., and)a light (L) chain variable region (abbreviated herein as VL). In someembodiments, an antibody comprises a constant domain, e.g., an Fcregion. An immunoglobulin constant domain refers to a heavy or lightchain constant domain. Human IgG heavy chain and light chain constantdomain amino acid sequences and their functional variations are known.With respect to the heavy chain, in some embodiments, the heavy chain ofan antibody described herein can be an alpha (a), delta (A), epsilon(c), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavychain of an antibody described herein can comprise a human alpha (a),delta (A), epsilon (c), gamma (γ) or mu (μ) heavy chain. In a particularembodiment, an antibody described herein comprises a human gamma 1 CH1,CH2, and/or (e.g., and) CH3 domain. In some embodiments, the amino acidsequence of the VH domain comprises the amino acid sequence of a humangamma (γ) heavy chain constant region, such as any known in the art.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra. In some embodiments, the VH domain comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or atleast 99% identical to any of the variable chain constant regionsprovided herein. In some embodiments, an antibody is modified, e.g.,modified via glycosylation, phosphorylation, sumoylation, and/or (e.g.,and) methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecule are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, an antibody is a constructthat comprises a polypeptide comprising one or more antigen bindingfragments of the disclosure linked to a linker polypeptide or animmunoglobulin constant domain. Linker polypeptides comprise two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Examples of linker polypeptides have beenreported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Still further, an antibody may be part of a larger immunoadhesionmolecule, formed by covalent or noncovalent association of the antibodyor antibody portion with one or more other proteins or peptides.Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,et al. (1994) Mol. Immunol. 31:1047-1058).

CDR: As used herein, the term “CDR” refers to the complementaritydetermining region within antibody variable sequences. A typicalantibody molecule comprises a heavy chain variable region (VH) and alight chain variable region (VL), which are usually involved in antigenbinding. The VH and VL regions can be further subdivided into regions ofhypervariability, also known as “complementarity determining regions”(“CDR”), interspersed with regions that are more conserved, which areknown as “framework regions” (“FR”). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The extent of the framework region and CDRs can be preciselyidentified using methodology known in the art, for example, by the Kabatdefinition, the IMGT definition, the Chothia definition, the AbMdefinition, and/or (e.g., and) the contact definition, all of which arewell known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; IMGT®, theinternational ImMunoGeneTics information System® http://www.imgt.org,Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M.et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., NucleicAcids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res.,31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006(2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic AcidsRes., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res.,37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res.,43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J.Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143(2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs. As used herein, aCDR may refer to the CDR defined by any method known in the art. Twoantibodies having the same CDR means that the two antibodies have thesame amino acid sequence of that CDR as determined by the same method,for example, the IMGT definition.

There are three CDRs in each of the variable regions of the heavy chainand the light chain, which are designated CDR1, CDR2 and CDR3, for eachof the variable regions. The term “CDR set” as used herein refers to agroup of three CDRs that occur in a single variable region capable ofbinding the antigen. The exact boundaries of these CDRs have beendefined differently according to different systems. The system describedby Kabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Sub-portions of CDRs may be designated as L1, L2 and L3 orH1, H2 and H3 where the “L” and the “H” designates the light chain andthe heavy chains regions, respectively. These regions may be referred toas Chothia CDRs, which have boundaries that overlap with Kabat CDRs.Other boundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems. Examples of CDR definition systemsare provided in Table 1.

TABLE 1 CDR Definitions IMGT¹ Kabat² Chothia³ CDR-H1 27-38 31-35 26-32CDR-H2 56-65 50-65 53-55 CDR-H3    105-116/117  95-102  96-101 CDR-L127-38 24-34 26-32 CDR-L2 56-65 50-56 50-52 CDR-L3    105-116/117 89-9791-96 ¹IMGT ®, the international ImMunoGeneTics information system ®,imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27: 209-212 (1999)²Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242 ³Chothia et al., J. Mol. Biol. 196: 901-917(1987))

CDR-grafted antibody: The term “CDR-grafted antibody” refers toantibodies which comprise heavy and light chain variable regionsequences from one species but in which the sequences of one or more ofthe CDR regions of VH and/or (e.g., and) VL are replaced with CDRsequences of another species, such as antibodies having murine heavy andlight chain variable regions in which one or more of the murine CDRs(e.g., CDR3) has been replaced with human CDR sequences.

Chimeric antibody: The term “chimeric antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions.

Complementary: As used herein, the term “complementary” refers to thecapacity for precise pairing between two nucleotides or two sets ofnucleotides. In particular, complementary is a term that characterizesan extent of hydrogen bond pairing that brings about binding between twonucleotides or two sets of nucleotides. For example, if a base at oneposition of an oligonucleotide is capable of hydrogen bonding with abase at the corresponding position of a target nucleic acid (e.g., anmRNA), then the bases are considered to be complementary to each otherat that position. Base pairings may include both canonical Watson-Crickbase pairing and non-Watson-Crick base pairing (e.g., Wobble basepairing and Hoogsteen base pairing). For example, in some embodiments,for complementary base pairings, adenosine-type bases (A) arecomplementary to thymidine-type bases (T) or uracil-type bases (U), thatcytosine-type bases (C) are complementary to guanosine-type bases (G),and that universal bases such as 3-nitropyrrole or 5-nitroindole canhybridize to and are considered complementary to any A, C, U, or T.Inosine (I) has also been considered in the art to be a universal baseand is considered complementary to any A, C, U or T.

Conservative amino acid substitution: As used herein, a “conservativeamino acid substitution” refers to an amino acid substitution that doesnot alter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Variants can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.Conservative substitutions of amino acids include substitutions madeamongst amino acids within the following groups: (a) M, I, L, V; (b) F,Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Covalently linked: As used herein, the term “covalently linked” refersto a characteristic of two or more molecules being linked together viaat least one covalent bond. In some embodiments, two molecules can becovalently linked together by a single bond, e.g., a disulfide bond ordisulfide bridge, that serves as a linker between the molecules.However, in some embodiments, two or more molecules can be covalentlylinked together via a molecule that serves as a linker that joins thetwo or more molecules together through multiple covalent bonds. In someembodiments, a linker may be a cleavable linker. However, in someembodiments, a linker may be a non-cleavable linker.

Cross-reactive: As used herein and in the context of a targeting agent(e.g., antibody), the term “cross-reactive,” refers to a property of theagent being capable of specifically binding to more than one antigen ofa similar type or class (e.g., antigens of multiple homologs, paralogs,or orthologs) with similar affinity or avidity. For example, in someembodiments, an antibody that is cross-reactive against human andnon-human primate antigens of a similar type or class (e.g., a humantransferrin receptor and non-human primate transferrin receptor) iscapable of binding to the human antigen and non-human primate antigenswith a similar affinity or avidity. In some embodiments, an antibody iscross-reactive against a human antigen and a rodent antigen of a similartype or class. In some embodiments, an antibody is cross-reactiveagainst a rodent antigen and a non-human primate antigen of a similartype or class. In some embodiments, an antibody is cross-reactiveagainst a human antigen, a non-human primate antigen, and a rodentantigen of a similar type or class.

DMD: As used herein, the term “DMD” refers to a gene that encodesdystrophin protein, a key component of the dystrophin-glycoproteincomplex, which bridges the inner cytoskeleton and the extracellularmatrix in muscle cells, particularly muscle fibers. Deletions,duplications, and point mutations in DMD may cause dystrophinopathies,such as Duchenne muscular dystrophy, Becker muscular dystrophy, orcardiomyopathy (e.g., DMD-associated dilated cardiomyopathy).Alternative promoter usage and alternative splicing result in numerousdistinct transcript variants and protein isoforms for this gene. In someembodiments, a dystrophin gene may be a human (Gene ID: 1756), non-humanprimate (e.g., Gene ID: 465559), or rodent gene (e.g., Gene ID: 13405;Gene ID: 24907). In addition, multiple human transcript variants (e.g.,as annotated under GenBank RefSeq Accession Numbers: NM_000109.3,NM_004006.2 (SEQ ID NO: 2239), NM_004009.3, NM_004010.3 and NM_004011.3)have been characterized that encode different protein isoforms.

DMD allele: As used herein, the term “DMD allele” refers to any one ofalternative forms (e.g., wild-type or mutant forms) of a DMD gene. Insome embodiments, a DMD allele may encode for dystrophin that retainsits normal and typical functions. In some embodiments, a DMD allele maycomprise one or more mutations that results in muscular dystrophy.Common mutations that lead to Duchenne muscular dystrophy involveframeshift, deletion, substitution, and duplicative mutations of one ormore of 79 exons present in a dystrophin allele, e.g., exon 8, exon 23,exon 41, exon 44, exon 50, exon 51, exon 52, exon 53, or exon 55.Further examples of DMD mutations are disclosed, for example, inFlanigan K M, et al., Mutational spectrum of DMD mutations indystrophinopathy patients: application of modern diagnostic techniquesto a large cohort. Hum Mutat. 2009 December; 30 (12):1657-66, thecontents of which are incorporated herein by reference in its entirety.

Dystrophinopathy: As used herein, the term “dystrophinopathy” refers toa muscle disease that results from one or more mutated DMD alleles.Dystrophinopathies include a spectrum of conditions (ranging from mildto severe) that includes Duchenne muscular dystrophy, Becker musculardystrophy, and DMD-associated dilated cardiomyopathy (DCM). In someembodiments, at one end of the spectrum, dystrophinopathy isphenotypically associated with an asymptomatic increase in serumconcentration of creatine phosphokinase (CK) and/or (e.g., and) musclecramps with myoglobinuria. In some embodiments, at the other end of thespectrum, dystrophinopathy is phenotypically associated with progressivemuscle diseases that are generally classified as Duchenne or Beckermuscular dystrophy when skeletal muscle is primarily affected and asDMD-associated dilated cardiomyopathy (DCM) when the heart is primarilyaffected. Symptoms of Duchenne muscular dystrophy include muscle loss ordegeneration, diminished muscle function, pseudohypertrophy of thetongue and calf muscles, higher risk of neurological abnormalities, anda shortened lifespan. Duchenne muscular dystrophy is associated withOnline Mendelian Inheritance in Man (OMIM) Entry #310200. Beckermuscular dystrophy is associated with OMIM Entry #300376. Dilatedcardiomyopathy is associated with OMIM Entry X #302045.

Exonic splicing enhancer (ESE): As used herein, the term “exonicsplicing enhancer” or “ESE” refers to a nucleic acid sequence motifwithin an exon of a gene, pre-mRNA, or mRNA that directs or enhancessplicing of pre-mRNA into mRNA, e.g., as described in Blencowe et al.,Trends Biochem Sci 25, 106-10. (2000), incorporated herein by reference.ESEs may direct or enhance splicing, for example, to remove one or moreintrons and/or one or more exons from a gene transcript. ESE motifs aretypically 6-8 nucleobases in length. SR proteins (e.g., proteins encodedby the gene SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8,SRSF9, SRSF10, SRSF11, SRSF12, TRA2A or TRA2B) bind to ESEs throughtheir RNA recognition motif region to facilitate splicing. ESE motifscan be identified through a number of methods, including those describedin Cartegni et al., Nucleic Acids Research, 2003, Vol. 31, No. 13,3568-3571, incorporated herein by reference.

Framework: As used herein, the term “framework” or “framework sequence”refers to the remaining sequences of a variable region minus the CDRs.Because the exact definition of a CDR sequence can be determined bydifferent systems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FRs within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region. Human heavychain and light chain acceptor sequences are known in the art. In oneembodiment, the acceptor sequences known in the art may be used in theantibodies disclosed herein.

Human antibody: The term “human antibody”, as used herein, is intendedto include antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibodies of thedisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

Humanized antibody: The term “humanized antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or (e.g., and) VL sequence has been altered to be more“human-like”, i.e., more similar to human germline variable sequences.One type of humanized antibody is a CDR-grafted antibody, in which humanCDR sequences are introduced into non-human VH and VL sequences toreplace the corresponding nonhuman CDR sequences. In one embodiment,humanized anti-transferrin receptor antibodies and antigen bindingportions are provided. Such antibodies may be generated by obtainingmurine anti-transferrin receptor monoclonal antibodies using traditionalhybridoma technology followed by humanization using in vitro geneticengineering, such as those disclosed in Kasaian et al PCT publicationNo. WO 2005/123126 A2.

Internalizing cell surface receptor: As used herein, the term,“internalizing cell surface receptor” refers to a cell surface receptorthat is internalized by cells, e.g., upon external stimulation, e.g.,ligand binding to the receptor. In some embodiments, an internalizingcell surface receptor is internalized by endocytosis. In someembodiments, an internalizing cell surface receptor is internalized byclathrin-mediated endocytosis. However, in some embodiments, aninternalizing cell surface receptor is internalized by aclathrin-independent pathway, such as, for example, phagocytosis,macropinocytosis, caveolae- and raft-mediated uptake or constitutiveclathrin-independent endocytosis. In some embodiments, the internalizingcell surface receptor comprises an intracellular domain, a transmembranedomain, and/or (e.g., and) an extracellular domain, which may optionallyfurther comprise a ligand-binding domain. In some embodiments, a cellsurface receptor becomes internalized by a cell after ligand binding. Insome embodiments, a ligand may be a muscle-targeting agent or amuscle-targeting antibody. In some embodiments, an internalizing cellsurface receptor is a transferrin receptor.

Isolated antibody: An “isolated antibody”, as used herein, is intendedto refer to an antibody that is substantially free of other antibodieshaving different antigenic specificities (e.g., an isolated antibodythat specifically binds transferrin receptor is substantially free ofantibodies that specifically bind antigens other than transferrinreceptor). An isolated antibody that specifically binds transferrinreceptor complex may, however, have cross-reactivity to other antigens,such as transferrin receptor molecules from other species. Moreover, anisolated antibody may be substantially free of other cellular materialand/or (e.g., and) chemicals.

Kabat numbering: The terms “Kabat numbering”, “Kabat definitions and“Kabat labeling” are used interchangeably herein. These terms, which arerecognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or an antigen binding portion thereof (Kabat et al. (1971)Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).For the heavy chain variable region, the hypervariable region rangesfrom amino acid positions 31 to 35 for CDR1, amino acid positions 50 to65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the lightchain variable region, the hypervariable region ranges from amino acidpositions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, andamino acid positions 89 to 97 for CDR3.

Molecular payload: As used herein, the term “molecular payload” refersto a molecule or species that functions to modulate a biologicaloutcome. In some embodiments, a molecular payload is linked to, orotherwise associated with a muscle-targeting agent. In some embodiments,the molecular payload is a small molecule, a protein, a peptide, anucleic acid, or an oligonucleotide. In some embodiments, the molecularpayload functions to modulate the transcription of a DNA sequence, tomodulate the expression of a protein, or to modulate the activity of aprotein. In some embodiments, the molecular payload is anoligonucleotide that comprises a strand having a region ofcomplementarity to a target gene.

Muscle-targeting agent: As used herein, the term, “muscle-targetingagent,” refers to a molecule that specifically binds to an antigenexpressed on muscle cells. The antigen in or on muscle cells may be amembrane protein, for example an integral membrane protein or aperipheral membrane protein. Typically, a muscle-targeting agentspecifically binds to an antigen on muscle cells that facilitatesinternalization of the muscle-targeting agent (and any associatedmolecular payload) into the muscle cells. In some embodiments, amuscle-targeting agent specifically binds to an internalizing, cellsurface receptor on muscles and is capable of being internalized intomuscle cells through receptor mediated internalization. In someembodiments, the muscle-targeting agent is a small molecule, a protein,a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In someembodiments, the muscle-targeting agent is linked to a molecularpayload.

Muscle-targeting antibody: As used herein, the term, “muscle-targetingantibody,” refers to a muscle-targeting agent that is an antibody thatspecifically binds to an antigen found in or on muscle cells. In someembodiments, a muscle-targeting antibody specifically binds to anantigen on muscle cells that facilitates internalization of themuscle-targeting antibody (and any associated molecular payment) intothe muscle cells. In some embodiments, the muscle-targeting antibodyspecifically binds to an internalizing, cell surface receptor present onmuscle cells. In some embodiments, the muscle-targeting antibody is anantibody that specifically binds to a transferrin receptor.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to anoligomeric nucleic acid compound of up to 200 nucleotides in length.Examples of oligonucleotides include, but are not limited to, RNAioligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers,phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guidenucleic acids (e.g., Cas9 guide RNAs), etc. Oligonucleotides may besingle-stranded or double-stranded. In some embodiments, anoligonucleotide may comprise one or more modified nucleotides (e.g.2′-O-methyl sugar modifications, purine or pyrimidine modifications). Insome embodiments, an oligonucleotide may comprise one or more modifiedinternucleotide linkage. In some embodiments, an oligonucleotide maycomprise one or more phosphorothioate linkages, which may be in the Rpor Sp stereochemical conformation.

Recombinant antibody: The term “recombinant human antibody”, as usedherein, is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described in more details in this disclosure), antibodies isolatedfrom a recombinant, combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002)Clin. Biochem. 35:425-445; Gavilondo J. V, and Larrick J. W. (2002)BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) ImmunologyToday 21:371-378), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., andGreen L. L. (2002) Current Opinion in Biotechnology 13:593-597; LittleM. et al (2000) Immunology Today 21:364-370) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo. One embodiment of the disclosure provides fully human antibodiescapable of binding human transferrin receptor which can be generatedusing techniques well known in the art, such as, but not limited to,using human Ig phage libraries such as those disclosed in Jermutus etal., PCT publication No. WO 2005/007699 A2.

Region of complementarity: As used herein, the term “region ofcomplementarity” refers to a nucleotide sequence, e.g., of aoligonucleotide, that is sufficiently complementary to a cognatenucleotide sequence, e.g., of a target nucleic acid, such that the twonucleotide sequences are capable of annealing to one another underphysiological conditions (e.g., in a cell). In some embodiments, aregion of complementarity is fully complementary to a cognate nucleotidesequence of target nucleic acid. However, in some embodiments, a regionof complementarity is partially complementary to a cognate nucleotidesequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99%complementarity). In some embodiments, a region of complementaritycontains 1, 2, 3, or 4 mismatches compared with a cognate nucleotidesequence of a target nucleic acid.

Specifically binds: As used herein, the term “specifically binds” refersto the ability of a molecule to bind to a binding partner with a degreeof affinity or avidity that enables the molecule to be used todistinguish the binding partner from an appropriate control in a bindingassay or other binding context. With respect to an antibody, the term,“specifically binds”, refers to the ability of the antibody to bind to aspecific antigen with a degree of affinity or avidity, compared with anappropriate reference antigen or antigens, that enables the antibody tobe used to distinguish the specific antigen from others, e.g., to anextent that permits preferential targeting to certain cells, e.g.,muscle cells, through binding to the antigen, as described herein. Insome embodiments, an antibody specifically binds to a target if theantibody has a K_(D) for binding the target of at least about 10⁻⁴ M,10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³M, or less. In some embodiments, an antibody specifically binds to thetransferrin receptor, e.g., an epitope of the apical domain oftransferrin receptor.

Subject: As used herein, the term “subject” refers to a mammal. In someembodiments, a subject is non-human primate, or rodent. In someembodiments, a subject is a human. In some embodiments, a subject is apatient, e.g., a human patient that has or is suspected of having adisease. In some embodiments, the subject is a human patient who has oris suspected of having a disease resulting from a mutated DMD genesequence, e.g., a mutation in an exon of a DMD gene sequence. In someembodiments, a subject has a dystrophinopathy, e.g., Duchenne musculardystrophy.

Transferrin receptor: As used herein, the term, “transferrin receptor”(also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalizingcell surface receptor that binds transferrin to facilitate iron uptakeby endocytosis. In some embodiments, a transferrin receptor may be ofhuman (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin.In addition, multiple human transcript variants have been characterizedthat encoded different isoforms of the receptor (e.g., as annotatedunder GenBank RefSeq Accession Numbers: NP_001121620.1, NP_003225.2,NP_001300894.1, and NP_001300895.1).

2′-modified nucleoside: As used herein, the terms “2′-modifiednucleoside” and “2′-modified ribonucleoside” are used interchangeablyand refer to a nucleoside having a sugar moiety modified at the 2′position. In some embodiments, the 2′-modified nucleoside is a 2′-4′bicyclic nucleoside, where the 2′ and 4′ positions of the sugar arebridged (e.g., via a methylene, an ethylene, or a (S)-constrained ethylbridge). In some embodiments, the 2′-modified nucleoside is anon-bicyclic 2′-modified nucleoside, e.g., where the 2′ position of thesugar moiety is substituted. Non-limiting examples of 2′-modifiednucleosides include: 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE),2′-O-N-methylacetamido (2′-O-NMA), locked nucleic acid (LNA,methylene-bridged nucleic acid), ethylene-bridged nucleic acid (ENA),and (S)-constrained ethyl-bridged nucleic acid (cEt). In someembodiments, the 2′-modified nucleosides described herein arehigh-affinity modified nucleotides and oligonucleotides comprising the2′-modified nucleotides have increased affinity to a target sequences,relative to an unmodified oligonucleotide. Examples of structures of2′-modified nucleosides are provided below:

II. Complexes

Provided herein are complexes that comprise a targeting agent, e.g. anantibody, covalently linked to a molecular payload. In some embodiments,a complex comprises a muscle-targeting antibody covalently linked to anoligonucleotide. A complex may comprise an antibody that specificallybinds a single antigenic site or that binds to at least two antigenicsites that may exist on the same or different antigens.

A complex may be used to modulate the activity or function of at leastone gene, protein, and/or (e.g., and) nucleic acid. In some embodiments,the molecular payload present with a complex is responsible for themodulation of a gene, protein, and/or (e.g., and) nucleic acids. Amolecular payload may be a small molecule, protein, nucleic acid,oligonucleotide, or any molecular entity capable of modulating theactivity or function of a gene, protein, and/or (e.g., and) nucleic acidin a cell. In some embodiments, a molecular payload is anoligonucleotide that targets a disease-associated repeat in musclecells.

In some embodiments, a complex comprises a muscle-targeting agent, e.g.an anti-transferrin receptor antibody, covalently linked to a molecularpayload, e.g. a mixmer antisense oligonucleotide that targets a mutatedDMD allele to promote exon skipping.

A. Muscle-Targeting Agents

Some aspects of the disclosure provide muscle-targeting agents, e.g.,for delivering a molecular payload to a muscle cell. In someembodiments, such muscle-targeting agents are capable of binding to amuscle cell, e.g., via specifically binding to an antigen on the musclecell, and delivering an associated molecular payload to the muscle cell.In some embodiments, the molecular payload is bound (e.g., covalentlybound) to the muscle targeting agent and is internalized into the musclecell upon binding of the muscle targeting agent to an antigen on themuscle cell, e.g., via endocytosis. It should be appreciated thatvarious types of muscle-targeting agents may be used in accordance withthe disclosure. For example, the muscle-targeting agent may comprise, orconsist of, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., anantibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., apolysaccharide). Exemplary muscle-targeting agents are described infurther detail herein, however, it should be appreciated that theexemplary muscle-targeting agents provided herein are not meant to belimiting.

Some aspects of the disclosure provide muscle-targeting agents thatspecifically bind to an antigen on muscle, such as skeletal muscle,smooth muscle, or cardiac muscle. In some embodiments, any of themuscle-targeting agents provided herein bind to (e.g., specifically bindto) an antigen on a skeletal muscle cell, a smooth muscle cell, and/or(e.g., and) a cardiac muscle cell.

By interacting with muscle-specific cell surface recognition elements(e.g., cell membrane proteins), both tissue localization and selectiveuptake into muscle cells can be achieved. In some embodiments, moleculesthat are substrates for muscle uptake transporters are useful fordelivering a molecular payload into muscle tissue. Binding to musclesurface recognition elements followed by endocytosis can allow evenlarge molecules such as antibodies to enter muscle cells. As anotherexample molecular payloads conjugated to transferrin or anti-transferrinreceptor antibodies can be taken up by muscle cells via binding totransferrin receptor, which may then be endocytosed, e.g., viaclathrin-mediated endocytosis.

The use of muscle-targeting agents may be useful for concentrating amolecular payload (e.g., oligonucleotide) in muscle while reducingtoxicity associated with effects in other tissues. In some embodiments,the muscle-targeting agent concentrates a bound molecular payload inmuscle cells as compared to another cell type within a subject. In someembodiments, the muscle-targeting agent concentrates a bound molecularpayload in muscle cells (e.g., skeletal, smooth, or cardiac musclecells) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than an amount innon-muscle cells (e.g., liver, neuronal, blood, or fat cells). In someembodiments, a toxicity of the molecular payload in a subject is reducedby at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered tothe subject when bound to the muscle-targeting agent.

In some embodiments, to achieve muscle selectivity, a muscle recognitionelement (e.g., a muscle cell antigen) may be required. As one example, amuscle-targeting agent may be a small molecule that is a substrate for amuscle-specific uptake transporter. As another example, amuscle-targeting agent may be an antibody that enters a muscle cell viatransporter-mediated endocytosis. As another example, a muscle targetingagent may be a ligand that binds to cell surface receptor on a musclecell. It should be appreciated that while transporter-based approachesprovide a direct path for cellular entry, receptor-based targeting mayinvolve stimulated endocytosis to reach the desired site of action.

i. Muscle-Targeting Antibodies

In some embodiments, the muscle-targeting agent is an antibody.Generally, the high specificity of antibodies for their target antigenprovides the potential for selectively targeting muscle cells (e.g.,skeletal, smooth, and/or (e.g., and) cardiac muscle cells). Thisspecificity may also limit off-target toxicity. Examples of antibodiesthat are capable of targeting a surface antigen of muscle cells havebeen reported and are within the scope of the disclosure. For example,antibodies that target the surface of muscle cells are described inArahata K., et al. “Immunostaining of skeletal and cardiac musclesurface membrane with antibody against Duchenne muscular dystrophypeptide” Nature 1988; 333: 861-3; Song K. S., et al. “Expression ofcaveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 isa component of the sarcolemma and co-fractionates with dystrophin anddystrophin-associated glycoproteins” J Biol Chem 1996; 271: 15160-5; andWeisbart R. H. et al., “Cell type specific targeted intracellulardelivery into muscle of a monoclonal antibody that binds myosin IIb” MolImmunol. 2003 March, 39(13):78309; the entire contents of each of whichare incorporated herein by reference.

a. Anti-Transferrin Receptor Antibodies

Some aspects of the disclosure are based on the recognition that agentsbinding to transferrin receptor, e.g., anti-transferrin-receptorantibodies, are capable of targeting muscle cell. Transferrin receptorsare internalizing cell surface receptors that transport transferrinacross the cellular membrane and participate in the regulation andhomeostasis of intracellular iron levels. Some aspects of the disclosureprovide transferrin receptor binding proteins, which are capable ofbinding to transferrin receptor. Accordingly, aspects of the disclosureprovide binding proteins (e.g., antibodies) that bind to transferrinreceptor. In some embodiments, binding proteins that bind to transferrinreceptor are internalized, along with any bound molecular payload, intoa muscle cell. As used herein, an antibody that binds to a transferrinreceptor may be referred to interchangeably as an, transferrin receptorantibody, an anti-transferrin receptor antibody, or an anti-TfRantibody. Antibodies that bind, e.g. specifically bind, to a transferrinreceptor may be internalized into the cell, e.g. throughreceptor-mediated endocytosis, upon binding to a transferrin receptor.

It should be appreciated that anti-transferrin receptor antibodies maybe produced, synthesized, and/or (e.g., and) derivatized using severalknown methodologies, e.g. library design using phage display. Exemplarymethodologies have been characterized in the art and are incorporated byreference (Diez, P. et al. “High-throughput phage-display screening inarray format”, Enzyme and microbial technology, 2015, 79, 34-41.;Christoph M. H. and Stanley, J. R. “Antibody Phage Display: Techniqueand Applications” J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.)“Human Hybridomas and Monoclonal Antibodies.” 1985, Springer.). In otherembodiments, an anti-transferrin antibody has been previouslycharacterized or disclosed. Antibodies that specifically bind totransferrin receptor are known in the art (see, e.g. U.S. Pat. No.4,364,934, filed Dec. 4, 1979, “Monoclonal antibody to a human earlythymocyte antigen and methods for preparing same”; U.S. Pat. No.8,409,573, filed Jun. 14, 2006, “Anti-CD71 monoclonal antibodies anduses thereof for treating malignant tumor cells”; U.S. Pat. No.9,708,406, filed May 20, 2014, “Anti-transferrin receptor antibodies andmethods of use”; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, “Lowaffinity blood brain barrier receptor antibodies and uses therefor”; WO2015/098989, filed Dec. 24, 2014, “Novel anti-Transferrin receptorantibody that passes through blood-brain barrier”; Schneider C. et al.“Structural features of the cell surface receptor for transferrin thatis recognized by the monoclonal antibody OKT9.” J Biol Chem. 1982,257:14, 8516-8522.; Lee et al. “Targeting Rat Anti-Mouse TransferrinReceptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”2000, J Pharmacol. Exp. Ther., 292: 1048-1052.).

Provided herein, in some aspects, are new anti-TfR antibodies for use asthe muscle targeting agents (e.g., in muscle targeting complexes). Insome embodiments, the anti-TfR antibody described herein binds totransferrin receptor with high specificity and affinity. In someembodiments, the anti-TfR antibody described herein specifically bindsto any extracellular epitope of a transferrin receptor or an epitopethat becomes exposed to an antibody. In some embodiments, anti-TfRantibodies provided herein bind specifically to transferrin receptorfrom human, non-human primates, mouse, rat, etc. In some embodiments,anti-TfR antibodies provided herein bind to human transferrin receptor.In some embodiments, the anti-TfR antibody described herein binds to anamino acid segment of a human or non-human primate transferrin receptor,as provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfRantibody described herein binds to an amino acid segment correspondingto amino acids 90-96 of a human transferrin receptor as set forth in SEQID NO: 105, which is not in the apical domain of the transferrinreceptor.

An example human transferrin receptor amino acid sequence, correspondingto NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1,Homo sapiens) is as follows:

(SEQ ID NO: 105) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALS GDVWDIDNEF.

An example non-human primate transferrin receptor amino acid sequence,corresponding to NCBI sequence NP_001244232.1 (transferrin receptorprotein 1, Macaca mulatta) is as follows:

(SEQ ID NO: 106) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALS GDVWDIDNEF

An example non-human primate transferrin receptor amino acid sequence,corresponding to NCBI sequence XP_005545315.1 (transferrin receptorprotein 1, Macaca fascicularis) is as follows:

(SEQ ID NO: 107)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF.

An example mouse transferrin receptor amino acid sequence, correspondingto NCBI sequence NP 001344227.1 (transferrin receptor protein 1, Musmusculus) is as follows:

(SEQ ID NO: 108)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF

In some embodiments, an anti-transferrin receptor antibody binds to anamino acid segment of the receptor as follows:FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the bindinginteractions between transferrin receptors and transferrin and/or (e.g.,and) human hemochromatosis protein (also known as HFE). In someembodiments, the anti-transferrin receptor antibody described hereindoes not bind an epitope in SEQ ID NO: 109.

Appropriate methodologies may be used to obtain and/or (e.g., and)produce antibodies, antibody fragments, or antigen-binding agents, e.g.,through the use of recombinant DNA protocols. In some embodiments, anantibody may also be produced through the generation of hybridomas (see,e.g., Kohler, G and Milstein, C. “Continuous cultures of fused cellssecreting antibody of predefined specificity” Nature, 1975, 256:495-497). The antigen-of-interest may be used as the immunogen in anyform or entity, e.g., recombinant or a naturally occurring form orentity. Hybridomas are screened using standard methods, e.g. ELISAscreening, to find at least one hybridoma that produces an antibody thattargets a particular antigen. Antibodies may also be produced throughscreening of protein expression libraries that express antibodies, e.g.,phage display libraries. Phage display library design may also be used,in some embodiments, (see, e.g. U.S. Pat. No. 5,223,409, filed Mar. 1,1991, “Directed evolution of novel binding proteins”; WO 1992/18619,filed Apr. 10, 1992, “Heterodimeric receptor libraries using phagemids”;WO 1991/17271, filed May 1, 1991, “Recombinant library screeningmethods”; WO 1992/20791, filed May 15, 1992, “Methods for producingmembers of specific binding pairs”; WO 1992/15679, filed Feb. 28, 1992,and “Improved epitope displaying phage”). In some embodiments, anantigen-of-interest may be used to immunize a non-human animal, e.g., arodent or a goat. In some embodiments, an antibody is then obtained fromthe non-human animal, and may be optionally modified using a number ofmethodologies, e.g., using recombinant DNA techniques. Additionalexamples of antibody production and methodologies are known in the art(see, e.g. Harlow et al. “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory, 1988.).

In some embodiments, an antibody is modified, e.g., modified viaglycosylation, phosphorylation, sumoylation, and/or (e.g., and)methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecules are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, there are about 1-10, about1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. Insome embodiments, a glycosylated antibody is fully or partiallyglycosylated. In some embodiments, an antibody is glycosylated bychemical reactions or by enzymatic means. In some embodiments, anantibody is glycosylated in vitro or inside a cell, which may optionallybe deficient in an enzyme in the N- or O-glycosylation pathway, e.g. aglycosyltransferase. In some embodiments, an antibody is functionalizedwith sugar or carbohydrate molecules as described in InternationalPatent Application Publication WO2014065661, published on May 1, 2014,entitled, “Modified antibody, antibody-conjugate and process for thepreparation thereof”.

In some embodiments, the anti-TfR antibody of the present disclosurecomprises a VL domain and/or (e.g., and) VH domain of any one of theanti-TfR antibodies selected from Table 2, and comprises a constantregion comprising the amino acid sequences of the constant regions of anIgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a andIgG2b) of immunoglobulin molecule. Non-limiting examples of humanconstant regions are described in the art, e.g., see Kabat E A et al.,(1991) supra.

In some embodiments, agents binding to transferrin receptor, e.g.,anti-TfR antibodies, are capable of targeting muscle cell and/or (e.g.,and) mediate the transportation of an agent across the blood brainbarrier. Transferrin receptors are internalizing cell surface receptorsthat transport transferrin across the cellular membrane and participatein the regulation and homeostasis of intracellular iron levels. Someaspects of the disclosure provide transferrin receptor binding proteins,which are capable of binding to transferrin receptor. Antibodies thatbind, e.g. specifically bind, to a transferrin receptor may beinternalized into the cell, e.g. through receptor-mediated endocytosis,upon binding to a transferrin receptor.

Provided herein, in some aspects, are humanized antibodies that bind totransferrin receptor with high specificity and affinity. In someembodiments, the humanized anti-TfR antibody described hereinspecifically binds to any extracellular epitope of a transferrinreceptor or an epitope that becomes exposed to an antibody. In someembodiments, the humanized anti-TfR antibodies provided herein bindspecifically to transferrin receptor from human, non-human primates,mouse, rat, etc. In some embodiments, the humanized anti-TfR antibodiesprovided herein bind to human transferrin receptor. In some embodiments,the humanized anti-TfR antibody described herein binds to an amino acidsegment of a human or non-human primate transferrin receptor, asprovided in SEQ ID NOs: 105-108. In some embodiments, the humanizedanti-TfR antibody described herein binds to an amino acid segmentcorresponding to amino acids 90-96 of a human transferrin receptor asset forth in SEQ ID NO: 105, which is not in the apical domain of thetransferrin receptor. In some embodiments, the humanized anti-TfRantibodies described herein binds to TfR1 but does not bind to TfR2.

In some embodiments, an anti-TFR antibody specifically binds a TfR1(e.g., a human or non-human primate TfR1) with binding affinity (e.g.,as indicated by Kd) of at least about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, or less. In someembodiments, the anti-TfR antibodies described herein binds to TfR1 witha KD of sub-nanomolar range. In some embodiments, the anti-TfRantibodies described herein selectively binds to transferrin receptor 1(TfR1) but do not bind to transferrin receptor 2 (TfR2). In someembodiments, the anti-TfR antibodies described herein binds to humanTfR1 and cyno TfR1 (e.g., with a Kd of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, or less), but does not bind to a mouse TfR1.The affinity and binding kinetics of the anti-TfR antibody can be testedusing any suitable method including but not limited to biosensortechnology (e.g., OCTET or BIACORE). In some embodiments, binding of anyone of the anti-TfR antibody described herein does not complete with orinhibit transferrin binding to the TfR1. In some embodiments, binding ofany one of the anti-TfR antibody described herein does not complete withor inhibit HFE-beta-2-microglobulin binding to the TfR1.

The anti-TfR antibodies described herein are humanized antibodies. TheCDR and variable region amino acid sequences of the mouse monoclonalanti-TfR antibody from which the humanized anti-TfR antibodies describedherein are derived are provided in Table 2.

TABLE 2 Mouse Monoclonal Anti-TfR Antibodies No. System Ab IMGT KabatChothia 3-A4 CDR- GFNIKDDY (SEQ ID NO: DDYMY (SEQ ID NO: 7)GFNIKDD (SEQ ID H1 1) NO: 12) CDR- IDPENGDT (SEQ ID NO:WIDPENGDTEYASKFQD ENG (SEQ ID NO: H2 2) (SEQ ID NO: 8) 13) CDR-TLWLRRGLDY (SEQ ID WLRRGLDY (SEQ ID NO: 9) LRRGLD (SEQ ID H3 NO: 3)NO: 14) CDR- KSLLHSNGYTY (SEQ ID RSSKSLLHSNGYTYLF (SEQ SKSLLHSNGYTY L1NO: 4) ID NO: 10) (SEQ ID NO: 15) CDR- RMS (SEQ ID NO: 5)RMSNLAS (SEQ ID NO: 11) RMS (SEQ ID NO: L2 5) CDR- MQHLEYPFT (SEQ IDMQHLEYPFT (SEQ ID NO: 6) HLEYPF (SEQ ID L3 NO: 6) NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPENGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 17) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18) 3-A4 CDR- GFNIKDDY (SEQ ID NO: DDYMY (SEQ ID NO: 7)GFNIKDD (SEQ ID N54T* H1 1) NO: 12) CDR- IDPETGDT (SEQ ID NO:WIDPETGDTEYASKFQD ETG (SEQ ID NO: H2 19) (SEQ ID NO: 20) 21) CDR-TLWLRRGLDY (SEQ ID WLRRGLDY (SEQ ID NO: 9) LRRGLD (SEQ ID H3 NO: 3)NO: 14) CDR- KSLLHSNGYTY (SEQ ID RSSKSLLHSNGYTYLF (SEQ SKSLLHSNGYTY L1NO: 4) ID NO: 10) (SEQ ID NO: 15) CDR- RMS (SEQ ID NO: 5)RMSNLAS (SEQ ID NO: 11) RMS(SEQ ID NO: L2 5) CDR- MQHLEYPFT (SEQ IDMQHLEYPFT (SEQ ID NO: 6) HLEYPF (SEQ ID L3 NO: 6) NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPETGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 22) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18) 3-A4 CDR- GFNIKDDY (SEQ ID NO: DDYMY (SEQ ID NO: 7)GFNIKDD (SEQ ID N54S* H1 1) NO: 12) CDR- IDPESGDT (SEQ ID NO:WIDPESGDTEYASKFQD ESG (SEQ ID NO: H2 23) (SEQ ID NO: 24) 25) CDR-TLWLRRGLDY (SEQ ID WLRRGLDY (SEQ ID NO: 9) LRRGLD (SEQ ID H3 NO: 3)NO: 14) CDR- KSLLHSNGYTY (SEQ ID RSSKSLLHSNGYTYLF (SEQ SKSLLHSNGYTY L1NO: 4) ID NO: 10) (SEQ ID NO: 15) CDR- RMS (SEQ ID NO: 5)RMSNLAS (SEQ ID NO: 11) RMS (SEQ ID NO: L2 5) CDR- MQHLEYPFT (SEQ IDMQHLEYPFT (SEQ ID NO: 6) HLEYPF (SEQ ID L3 NO: 6) NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPESGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 26) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18) 3-M12 CDR- GYSITSGYY (SEQ ID SGYYWN (SEQ ID NO: 33)GYSITSGY (SEQ H1 NO: 27) ID NO: 38) CDR- ITFDGAN (SEQ ID NO:YITFDGANNYNPSLKN (SEQ FDG (SEQ ID NO: H2 28) ID NO: 34) 39) CDR-TRSSYDYDVLDY (SEQ SSYDYDVLDY (SEQ ID NO: SYDYDVLD (SEQ H3 ID NO: 29) 35)ID NO: 40) CDR- QDISNF (SEQ ID NO: 30) RASQDISNFLN (SEQ ID NO:SQDISNF (SEQ ID L1 36) NO: 41) CDR- YTS (SEQ ID NO: 31)YTSRLHS (SEQ ID NO: 37) YTS (SEQ ID NO: L2 31) CDR- QQGHTLPYT (SEQ IDQQGHTLPYT (SEQ ID NO: 32) GHTLPY (SEQ ID L3 NO: 32) NO: 42) VHDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYITFDGANNYNPSLKNRISITRDTSKNQFFLKLTSVTTEDTATYYCTRSSYDYDVLDYWGQGTTLTVSS (SEQ ID NO: 43) VLDIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQRPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTVSNLEQEDIATYFCQQGHTLPYTFGGGTKLEIK (SEQ ID NO: 44)5-H12 CDR- GYSFTDYC (SEQ ID NO: DYCIN (SEQ ID NO: 51) GYSFTDY (SEQ ID H145) NO: 56) CDR- IYPGSGNT (SEQ ID NO: WIYPGSGNTRYSERFKG GSG (SEQ ID NO:H2 46) (SEQ ID NO: 52) 57) CDR- AREDYYPYHGMDY EDYYPYHGMDY (SEQ IDDYYPYHGMD H3 (SEQ ID NO: 47) NO: 53) (SEQ ID NO: 58) CDR-ESVDGYDNSF (SEQ ID RASESVDGYDNSFMH (SEQ SESVDGYDNSF Ll NO: 48)ID NO: 54) (SEQ ID NO: 59) CDR- RAS (SEQ ID NO: 49)RASNLES (SEQ ID NO: 55) RAS (SEQ ID NO: L2 49) CDR- QQSSEDPWT (SEQ IDQQSSEDPWT (SEQ ID NO: 50) SSEDPW (SEQ ID L3 NO: 50) NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYCINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 61) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62) 5-H12 CDR- GYSFTDYY (SEQ ID DYYIN (SEQ ID NO: 64)GYSFTDY (SEQ ID C33Y* H1 NO: 63) NO: 56) CDR- IYPGSGNT (SEQ ID NO:WIYPGSGNTRYSERFKG GSG (SEQ ID NO: H2 46) (SEQ ID NO: 52) 57) CDR-AREDYYPYHGMDY EDYYPYHGMDY (SEQ ID DYYPYHGMD H3 (SEQ ID NO: 47) NO: 53)(SEQ ID NO: 58) CDR- ESVDGYDNSF (SEQ ID RASESVDGYDNSFMH (SEQ SESVDGYDNSFL1 NO: 48) ID NO: 54) (SEQ ID NO: 59) CDR- RAS (SEQ ID NO: 49)RASNLES (SEQ ID NO: 55) RAS (SEQ ID NO: L2 49) CDR- QQSSEDPWT (SEQ IDQQSSEDPWT (SEQ ID NO: 50) SSEDPW (SEQ ID L3 NO: 50) NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYYINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 65) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62) 5-H12 CDR- GYSFTDYD (SEQ ID DYDIN (SEQ ID NO: 67)GYSFTDY (SEQ ID C33D* H1 NO: 66) NO: 56) CDR- IYPGSGNT (SEQ ID NO:WIYPGSGNTRYSERFKG GSG (SEQ ID NO: H2 46) (SEQ ID NO: 52) 57) CDR-AREDYYPYHGMDY EDYYPYHGMDY (SEQ ID DYYPYHGMD H3 (SEQ ID NO: 47) NO: 53)(SEQ ID NO: 58) CDR- ESVDGYDNSF (SEQ ID RASESVDGYDNSFMH (SEQ SESVDGYDNSFL1 NO: 48) ID NO: 54) (SEQ ID NO: 59) CDR- RAS (SEQ ID NO: 49)RASNLES (SEQ ID NO: 55) RAS (SEQ ID NO: L2 49) CDR- QQSSEDPWT (SEQ IDQQSSEDPWT (SEQ ID NO: 50) SSEDPW (SEQ ID L3 NO: 50) NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYDINWVNQRPGQGLEWIGWIYPGS GNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 68) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62) *mutation positions are according to Kabat numberingof the respective VH sequences containing the mutations

In some embodiments, the anti-TfR antibody of the present disclosure isa humanized variant of any one of the anti-TfR antibodies provided inTable 2. In some embodiments, the anti-TfR antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2,and a CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 in anyone of the anti-TfR antibodies provided in Table 2, and comprises ahumanized heavy chain variable region and/or (e.g., and) a humanizedlight chain variable region.

Humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some embodiments, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs derived from one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

Humanized antibodies and methods of making them are known, e.g., asdescribed in Almagro et al., Front. Biosci. 13:1619-1633 (2008);Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'lAcad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34(2005); Padlan et al., Mol. Immunol. 28:489-498 (1991); Dall'Acqua etal., Methods 36:43-60 (2005); Osbourn et al., Methods 36:61-68 (2005);and Klimka et al., Br. J. Cancer, 83:252-260 (2000), the contents of allof which are incorporated herein by reference. Human framework regionsthat may be used for humanization are described in e.g., Sims et al. J.Immunol. 151:2296 (1993); Carter et al., Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993); Almagro etal., Front. Biosci. 13:1619-1633 (2008)); Baca et al., J. Biol. Chem.272:10678-10684 (1997); and Rosok et al., J Biol. Chem. 271:22611-22618(1996), the contents of all of which are incorporated herein byreference.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising one or more amino acidvariations (e.g., in the VH framework region) as compared with any oneof the VHs listed in Table 2, and/or (e.g., and) a humanized VLcomprising one or more amino acid variations (e.g., in the VL frameworkregion) as compared with any one of the VLs listed in Table 2.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH containing no more than 25 aminoacid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VH of any ofthe anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs:17, 22, 26, 43, 61, 65, and 68). Alternatively or in addition (e.g., inaddition), the humanized anti-TfR antibody of the present disclosurecomprises a humanized VL containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL of any oneof the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ IDNOs: 18, 44, and 62).

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%)identical in the framework regions to the VH of any of the anti-TfRantibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26,43, 61, 65, and 68). Alternatively or in addition (e.g., in addition),In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VL comprising an amino acid sequencethat is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%)identical in the framework regions to the VL of any of the anti-TfRantibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and62).

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 1 (according to the IMGT definition system),a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19,or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3having the amino acid sequence of SEQ ID NO: 3 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VH as set forth in SEQ ID NO: 17,SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or in addition (e.g., inaddition), the anti-TfR antibody of the present disclosure comprises ahumanized VL comprising a CDR-L1 having the amino acid sequence of SEQID NO: 4 (according to the IMGT definition system), a CDR-L2 having theamino acid sequence of SEQ ID NO: 5 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6(according to the IMGT definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VL as setforth in SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 1 (according to the IMGT definition system),a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19,or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3having the amino acid sequence of SEQ ID NO: 3 (according to the IMGTdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VH as set forthin SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 4 (according to the IMGTdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 5 (according to the IMGT definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 6 (according to the IMGT definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VL as set forth in anyone of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 7 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), aCDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to theKabat definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VH as set forthin SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 10 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 11 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 7 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), aCDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to theKabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%,95%, 98%, or 99%) identical in the framework regions to the VH as setforth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternativelyor in addition (e.g., in addition), the humanized anti-TfR antibody ofthe present disclosure comprises a humanized VL comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 10 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 11 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin any one of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 12 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQID NO: 21, or SEQ ID NO: 25 (according to the Chothia definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14(according to the Chothia definition system), and containing no morethan 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid variation) in the framework regions as compared with the VHas set forth in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to theChothia definition system), a CDR-L2 having the amino acid sequence ofSEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 16 (according to theChothia definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL as set forthin SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 12 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQID NO: 21, or SEQ ID NO: 25 (according to the Chothia definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 22 or SEQID NO: 26. Alternatively or in addition (e.g., in addition), theanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15(according to the Chothia definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 5 (according to the Chothia definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in any one of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 27 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 29 (according to the IMGT definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to theIMGT definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 31 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 32 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 27 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 29 (according to the IMGT definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30(according to the IMGT definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 31 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32(according to the IMGT definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 33 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 35 (according to the Kabat definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to theKabat definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 37 (according to the Kabat definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 32 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 33 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 35 (according to the Kabat definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 36(according to the Kabat definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 37 (according to the Kabat definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32(according to the Kabat definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 38 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 40 (according to the Chothia definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to theChothia definition system), a CDR-L2 having the amino acid sequence ofSEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 42 (according to theChothia definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL as set forthin SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 38 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 40 (according to the Chothia definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41(according to the Chothia definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 31 (according to the Chothia definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66(according to the IMGT definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 46 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47(according to the IMGT definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VH as setforth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternativelyor in addition (e.g., in addition), the humanized anti-TfR antibody ofthe present disclosure comprises a humanized VL comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 48 (according to the IMGTdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 49 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66(according to the IMGT definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 46 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47(according to the IMGT definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to theIMGT definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 49 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the IMGTdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67(according to the Kabat definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 52 (according to the Kabat definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53(according to the Kabat definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VH as setforth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively orin addition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 54 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 55 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67(according to the Kabat definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 52 (according to the Kabat definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53(according to the Kabat definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to theKabat definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 55 (according to the Kabat definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 50 (according to the Kabatdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 56 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 58 (according to the Chothia definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 61, SEQ IDNO: 65, SEQ ID NO: 68. Alternatively or in addition (e.g., in addition),the humanized anti-TfR antibody of the present disclosure comprises ahumanized VL comprising a CDR-L1 having the amino acid sequence of SEQID NO: 59 (according to the Chothia definition system), a CDR-L2 havingthe amino acid sequence of SEQ ID NO: 49 (according to the Chothiadefinition system), and a CDR-L3 having the amino acid sequence of SEQID NO: 60 (according to the Chothia definition system), and containingno more than 25 amino acid variations (e.g., no more than 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acid variation) in the framework regions as compared withthe VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 56 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 58 (according to the Chothia definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively or in addition(e.g., in addition), the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VL comprising a CDR-L1 having the aminoacid sequence of SEQ ID NO: 59 (according to the Chothia definitionsystem), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49(according to the Chothia definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 60 (according to the Chothiadefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

Examples of amino acid sequences of the humanized anti-TfR antibodiesdescribed herein are provided in Table 3.

TABLE 3 Variable Regions of Humanized Anti-TfR Antibodies AntibodyVariable Region Amino Acid Sequence** 3A4 V_(H): VH3 (N54T*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 69) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3A4 V_(H): VH3 (N54S*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 71) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3A4 V_(H): VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 72) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3M12 V_(H): VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 73) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 74)3M12 V_(H): VH3/Vκ3QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 73) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 75)3M12 V_(H): VH4/Vκ2QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 76) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 74)3M12 V_(H): VH4/Vκ3QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 76) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 75)5H12 V_(H): VH5 (C33Y*)/Vκ3QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSS (SEQ ID NO: 77) V_(L):DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 78) 5H12 V_(H): VH5 (C33D*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSS (SEQ ID NO: 79) V_(L):DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 80) 5H12 V_(H): VH5 (C33Y*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSS (SEQ ID NO: 77) V_(L):DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 80) *mutation positions are according to Kabat numberingof the respective VH sequences containing the mutations **CDRs accordingto the Kabat numbering system are bolded

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising the CDR-H1, CDR-H2, andCDR-H3 of any one of the anti-TfR antibodies provided in Table 2 andcomprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)amino acid variations in the framework regions as compared with therespective humanized VH provided in Table 3. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising the CDR-L1,CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided inTable 2 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) amino acid variations in the framework regions as compared withthe respective humanized VL provided in Table 3.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 69, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 69 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 71, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 71 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 72, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 72 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 74. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 73 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 75. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 73 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 74. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 76 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 75. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 76 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 78. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 77 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 79, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 80. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 79 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 80. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 77 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the humanized anti-TfR antibody described herein isa full-length IgG, which can include a heavy constant region and a lightconstant region from a human antibody. In some embodiments, the heavychain of any of the anti-TfR antibodies as described herein maycomprises a heavy chain constant region (CH) or a portion thereof (e.g.,CH1, CH2, CH3, or a combination thereof). The heavy chain constantregion can of any suitable origin, e.g., human, mouse, rat, or rabbit.In one specific example, the heavy chain constant region is from a humanIgG (a gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of ahuman IgG1 constant region is given below:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

(SEQ ID NO: 81) YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  K

In some embodiments, the heavy chain of any of the anti-TfR antibodiesdescribed herein comprises a mutant human IgG1 constant region. Forexample, the introduction of LALA mutations (a mutant derived from mAbb12 that has been mutated to replace the lower hinge residues Leu234Leu235 with Ala234 and Ala235) in the CH2 domain of human IgG1 is knownto reduce Fcg receptor binding (Bruhns, P., et al. (2009) and Xu, D. etal. (2000)). The mutant human IgG1 constant region is provided below(mutations bonded and underlined):

(SEQ ID NO: 82) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

In some embodiments, the light chain of any of the anti-TfR antibodiesdescribed herein may further comprise a light chain constant region(CL), which can be any CL known in the art. In some examples, the CL isa kappa light chain. In other examples, the CL is a lambda light chain.In some embodiments, the CL is a kappa light chain, the sequence ofwhich is provided below:

(SEQ ID NO: 83) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL  SSPVTKSFNRGEC

Other antibody heavy and light chain constant regions are well known inthe art, e.g., those provided in the IMGT database (www.imgt.org) or atwww.vbase2.org/vbstat.php., both of which are incorporated by referenceherein.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising any one of the VH as listed in Table3 or any variants thereof and a heavy chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, thehumanized anti-TfR antibody described herein comprises a heavy chaincomprising any one of the VH as listed in Table 3 or any variantsthereof and a heavy chain constant region that contains no more than 25amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with SEQ ID NO: 81 or SEQ ID NO: 82. In someembodiments, the humanized anti-TfR antibody described herein comprisesa heavy chain comprising any one of the VH as listed in Table 3 or anyvariants thereof and a heavy chain constant region as set forth in SEQID NO: 81. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises heavy chain comprising any one of the VH aslisted in Table 3 or any variants thereof and a heavy chain constantregion as set forth in SEQ ID NO: 82.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a light chain comprising any one of the VL as listed in Table3 or any variants thereof and a light chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfRantibody described herein comprises a light chain comprising any one ofthe VL as listed in Table 3 or any variants thereof and a light chainconstant region contains no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withSEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises a light chain comprising any one of the VL aslisted in Table 3 or any variants thereof and a light chain constantregion set forth in SEQ ID NO: 83.

Examples of IgG heavy chain and light chain amino acid sequences of theanti-TfR antibodies described are provided in Table 4 below.

TABLE 4Heavy chain and light chain sequences of examples of humanized anti-TfR IgGsAntibody IgG Heavy Chain/Light Chain Sequences** 3A4Heavy Chain (with wild type human IgG1 constant region) VH3 (N54T*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRqPPGKGLEWIGWIDPETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with wild type human IgG1 constant region) VH3 (N54S*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with wild type human IgG1 constant region) VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 87)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3M12 Heavy Chain (with wild type human IgG1 constant region) VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with wild type human IgG1 constant region) VH3/Vκ3QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 3M12Heavy Chain (with wild type human IgG1 constant region) VH4/Vκ2QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with wild type human IgG1 constant region) VH4/Vκ3QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 5H12Heavy Chain (with wild type human IgG1 constant region) VH5 (C33Y*)/Vκ3QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)Light Chain (with kappa light chain constant region)DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93) 5H12Heavy Chain (with wild type human IgG1 constant region) VH5 (C33D*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)5H12 Heavy Chain (with wild type human IgG1 constant region)VH5 (C33Y*)/Vκ4 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)*mutation positions are according to Kabat numbering of the respectiveVH sequences containing the mutations **CDRs according to the Kabatnumbering system are bolded; VH/VL sequences underlined

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in any one ofSEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or in addition(e.g., in addition), the humanized anti-TfR antibody of the presentdisclosure comprises a light chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the light chain as set forth in any one ofSEQ ID NOs: 85, 89, 90, 93, and 95.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to anyone of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody describedherein comprises a light chain comprising an amino acid sequence that isat least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical toany one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, theanti-TfR antibody described herein comprises a heavy chain comprisingthe amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91,92, and 94. Alternatively or in addition (e.g., in addition), theanti-TfR antibody described herein comprises a light chain comprisingthe amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 84, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 86, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 86 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 87, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 93. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 94, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 94 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the anti-TfR antibody is a Fab fragment, Fab′fragment, or F(ab′)2 fragment of an intact antibody (full-lengthantibody). Antigen binding fragment of an intact antibody (full-lengthantibody) can be prepared via routine methods (e.g., recombinantly or bydigesting the heavy chain constant region of a full length IgG using anenzyme such as papain). For example, F(ab′)2 fragments can be producedby pepsin or papain digestion of an antibody molecule, and Fab′fragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments. In some embodiments, a heavy chain constant region ina Fab fragment of the anti-TfR1 antibody described herein comprises theamino acid sequence of:

(SEQ ID NO: 96) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHT 

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising any one of the VH as listed in Table3 or any variants thereof and a heavy chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 96. In some embodiments, the humanized anti-TfRantibody described herein comprises a heavy chain comprising any one ofthe VH as listed in Table 3 or any variants thereof and a heavy chainconstant region that contains no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) ascompared with SEQ ID NO: 96. In some embodiments, the humanized anti-TfRantibody described herein comprises a heavy chain comprising any one ofthe VH as listed in Table 3 or any variants thereof and a heavy chainconstant region as set forth in SEQ ID NO: 96.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a light chain comprising any one of the VL as listed in Table3 or any variants thereof and a light chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfRantibody described herein comprises a light chain comprising any one ofthe VL as listed in Table 3 or any variants thereof and a light chainconstant region contains no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withSEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises a light chain comprising any one of the VL aslisted in Table 3 or any variants thereof and a light chain constantregion set forth in SEQ ID NO: 83.

Examples of Fab heavy chain and light chain amino acid sequences of theanti-TfR antibodies described are provided in Table 5 below.

TABLE 5Heavy chain and light chain sequences of examples of humanized anti-TfR FabsAntibody Fab Heavy Chain/Light Chain Sequences** 3A4Heavy Chain (with partial human IgG1 constant region) VH3 (N54T*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 97)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with partial human IgG1 constant region) VH3 (N54S*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 98)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with partial human IgG1 constant region) VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 99)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3M12Heavy Chain (with partial human IgG1 constant region) VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 100)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89)3M12 Heavy Chain (with partial human IgG1 constant region) VH3/Vκ3QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 100)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90)3M12 Heavy Chain (with partial human IgG1 constant region) VH4/Vκ2QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 101)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89)3M12 Heavy Chain (with partial human IgG1 constant region) VH4/Vκ3QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 101)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90)5H12 Heavy Chain (with partial human IgG1 constant region)VH5 (C33Y*)/Vκ3 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 102)Light Chain (with kappa light chain constant region)DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93) 5H12Heavy Chain (with partial human IgG1 constant region) VH5 (C33D*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NQ: 103)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95) 5H12Heavy Chain (with partial human IgG1 constant region) VH5 (C33Y*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 102)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95) *mutation positions are according to Kabatnumbering of the respective VH sequences containing the mutations **CDRsaccording to the Kabat numbering system are bolded; VH/VL sequencesunderlined

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in any one ofSEQ ID NOs: 97-103. Alternatively or in addition (e.g., in addition),the humanized anti-TfR antibody of the present disclosure comprises alight chain containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withthe light chain as set forth in any one of SEQ ID NOs: 85, 89, 90, 93,and 95.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to anyone of SEQ ID NOs: 97-103. Alternatively or in addition (e.g., inaddition), the humanized anti-TfR antibody described herein comprises alight chain comprising an amino acid sequence that is at least 75%(e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfRantibody described herein comprises a heavy chain comprising the aminoacid sequence of any one of SEQ ID NOs: 97-103. Alternatively or inaddition (e.g., in addition), the anti-TfR antibody described hereincomprises a light chain comprising the amino acid sequence of any one ofSEQ ID NOs: 85, 89, 90, 93, and 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 97, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 97 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 98, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 98 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 99, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 99 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 100 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 100 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 93. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 103, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 103 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR receptor antibodiesdescribed herein can be in any antibody form, including, but not limitedto, intact (i.e., full-length) antibodies, antigen-binding fragmentsthereof (such as Fab, Fab′, F(ab′)2, Fv), single chain antibodies,bi-specific antibodies, or nanobodies. In some embodiments, humanizedthe anti-TfR antibody described herein is a scFv. In some embodiments,the humanized anti-TfR antibody described herein is a scFv-Fab (e.g.,scFv fused to a portion of a constant region). In some embodiments, theanti-TfR receptor antibody described herein is a scFv fused to aconstant region (e.g., human IgG1 constant region as set forth in SEQ IDNO: 81 or SEQ ID NO: 82, or a portion thereof such as the Fc portion) ateither the N-terminus of C-terminus.

In some embodiments, conservative mutations can be introduced intoantibody sequences (e.g., CDRs or framework sequences) at positionswhere the residues are not likely to be involved in interacting with atarget antigen (e.g., transferrin receptor), for example, as determinedbased on a crystal structure. In some embodiments, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of an anti-TfR antibody described herein (e.g., in a CH2 domain(residues 231-340 of human IgG1) and/or (e.g., and) CH3 domain (residues341-447 of human IgG1) and/or (e.g., and) the hinge region, withnumbering according to the Kabat numbering system (e.g., the EU index inKabat)) to alter one or more functional properties of the antibody, suchas serum half-life, complement fixation, Fc receptor binding and/or(e.g., and) antigen-dependent cellular cytotoxicity.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain can be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of a muscle-targetingantibody described herein (e.g., in a CH2 domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) CH3 domain (residues 341-447 of humanIgG1) and/or (e.g., and) the hinge region, with numbering according tothe Kabat numbering system (e.g., the EU index in Kabat)) to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to decrease the half-life of the anti-anti-TfRantibody in vivo. In some embodiments, one, two or more amino acidmutations (i.e., substitutions, insertions or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In some embodiments, the antibodies can haveone or more amino acid mutations (e.g., substitutions) in the secondconstant (CH2) domain (residues 231-340 of human IgG1) and/or (e.g.,and) the third constant (CH3) domain (residues 341-447 of human IgG1),with numbering according to the EU index in Kabat (Kabat E A et al.,(1991) supra). In some embodiments, the constant region of the IgG1 ofan antibody described herein comprises a methionine (M) to tyrosine (Y)substitution in position 252, a serine (S) to threonine (T) substitutionin position 254, and a threonine (T) to glutamic acid (E) substitutionin position 256, numbered according to the EU index as in Kabat. SeeU.S. Pat. No. 7,658,921, which is incorporated herein by reference. Thistype of mutant IgG, referred to as “YTE mutant” has been shown todisplay fourfold increased half-life as compared to wild-type versionsof the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, an antibody comprises an IgG constantdomain comprising one, two, three or more amino acid substitutions ofamino acid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU index as in Kabat.

In some embodiments, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the anti-anti-TfR antibody. The effector ligand to whichaffinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, thedeletion or inactivation (through point mutations or other means) of aconstant region domain can reduce Fc receptor binding of the circulatingantibody thereby increasing tumor localization. See, e.g., U.S. Pat.Nos. 5,585,097 and 8,591,886 for a description of mutations that deleteor inactivate the constant domain and thereby increase tumorlocalization. In some embodiments, one or more amino acid substitutionsmay be introduced into the Fc region of an antibody described herein toremove potential glycosylation sites on Fc region, which may reduce Fcreceptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino in the constant region of ananti-TfR antibody described herein can be replaced with a differentamino acid residue such that the antibody has altered Clq binding and/or(e.g., and) reduced or abolished complement dependent cytotoxicity(CDC). This approach is described in further detail in U.S. Pat. No.6,194,551 (Idusogie et al). In some embodiments, one or more amino acidresidues in the N-terminal region of the CH2 domain of an antibodydescribed herein are altered to thereby alter the ability of theantibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments, the Fcregion of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or (e.g., and) to increase the affinity of theantibody for an Fcγ receptor. This approach is described further inInternational Publication No. WO 00/42072.

In some embodiments, the heavy and/or (e.g., and) light chain variabledomain(s) sequence(s) of the antibodies provided herein can be used togenerate, for example, CDR-grafted, chimeric, humanized, or compositehuman antibodies or antigen-binding fragments, as described elsewhereherein. As understood by one of ordinary skill in the art, any variant,CDR-grafted, chimeric, humanized, or composite antibodies derived fromany of the antibodies provided herein may be useful in the compositionsand methods described herein and will maintain the ability tospecifically bind transferrin receptor, such that the variant,CDR-grafted, chimeric, humanized, or composite antibody has at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% or more binding to transferrin receptor relative to the originalantibody from which it is derived.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like hinge sequence. Accordingly, any of theantibodies may include a stabilizing ‘Adair’ mutation.

In some embodiments, an antibody is modified, e.g., modified viaglycosylation, phosphorylation, sumoylation, and/or (e.g., and)methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecules are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, there are about 1-10, about1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. Insome embodiments, a glycosylated antibody is fully or partiallyglycosylated. In some embodiments, an antibody is glycosylated bychemical reactions or by enzymatic means. In some embodiments, anantibody is glycosylated in vitro or inside a cell, which may optionallybe deficient in an enzyme in the N- or O-glycosylation pathway, e.g. aglycosyltransferase. In some embodiments, an antibody is functionalizedwith sugar or carbohydrate molecules as described in InternationalPatent Application Publication WO2014065661, published on May 1, 2014,entitled, “Modified antibody, antibody-conjugate and process for thepreparation thereof”.

In some embodiments, any one of the anti-TfR1 antibodies describedherein may comprise a signal peptide in the heavy and/or (e.g., and)light chain sequence (e.g., a N-terminal signal peptide). In someembodiments, the anti-TfR1 antibody described herein comprises any oneof the VH and VL sequences, any one of the IgG heavy chain and lightchain sequences, or any one of the Fab heavy chain and light chainsequences described herein, and further comprises a signal peptide(e.g., a N-terminal signal peptide). In some embodiments, the signalpeptide comprises the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ IDNO: 104).

Other Known Anti-Transferrin Receptor Antibodies

Any other appropriate anti-transferrin receptor antibodies known in theart may be used as the muscle-targeting agent in the complexes disclosedherein. Examples of known anti-transferrin receptor antibodies,including associated references and binding epitopes, are listed inTable 8. In some embodiments, the anti-transferrin receptor antibodycomprises the complementarity determining regions (CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrinreceptor antibodies provided herein, e.g., anti-transferrin receptorantibodies listed in Table 8.

TABLE 8 List of anti-transferrin receptor antibody clones, includingassociated references and binding epitope information. Antibody CloneName Reference(s) Epitope/Notes OKT9 U.S. Pat. No. 4,364,934, filed Dec.4, 1979, Apical domain of TfR entitled “MONOCLONAL ANTIBODY (residues305-366 of TO A HUMAN EARLY THYMOCYTE human TfR sequence ANTIGEN ANDMETHODS FOR XM_052730.3, PREPARING SAME” available in GenBank) SchneiderC. et al. “Structural features of the cell surface receptor fortransferrin that is recognized by the monoclonal antibody OKT9.” J BiolChem. 1982, 257: 14, 8516- 8522. (From JCR) WO 2015/098989, filed Dec.24, 2014, Apical domain Clone M11 “Novel anti-Transferrin receptor(residues 230-244 and Clone M23 antibody that passes through blood-326-347 of TfR) and Clone M27 brain barrier” protease-like domain CloneB84 U.S. Pat. No. 9,994,641, filed (residues 461-473) Dec. 24, 2014,“Novel anti-Transferrin receptor antibody that passes throughblood-brain barrier” (From WO 2016/081643, filed May 26, 2016, Apicaldomain and Genentech) entitled “ANTI-TRANSFERRIN non-apical regions 7A4,8A2, RECEPTOR ANTIBODIES AND 15D2, 10D11, METHODS OF USE” 7B10, 15G11,U.S. Pat. No. 9,708,406, filed 16G5, 13C3, May 20, 2014,“Anti-transferrin receptor 16G4, 16F6, antibodies and methods of use”7G7, 4C2, 1B12, and 13D4 (From Lee et al. “Targeting Rat Anti-MouseArmagen) Transferrin Receptor Monoclonal 8D3 Antibodies throughBlood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292:1048-1052. US Patent App. 2010/077498, filed Sep. 11, 2008, entitled“COMPOSITIONS AND METHODS FOR BLOOD- BRAIN BARRIER DELIVERY IN THEMOUSE” OX26 Haobam, B. et al. 2014. Rab17- mediated recycling endosomescontribute to autophagosome formation in response to Group AStreptococcus invasion. Cellular microbiology. 16: 1806-21. DF1513Ortiz-Zapater E et al. Trafficking of the human transferrin receptor inplant cells: effects of tyrphostin A23 and brefeldin A. Plant J 48:757-70 (2006). 1A1B2, 66IG10, Commercially available anti-transferrinNovus Biologicals MEM-189, JF0956, receptor antibodies. 8100 SouthparkWay, 29806, 1A1B2, A-8 Littleton CO TFRC/1818, 1E6, 80120 66Ig10,TFRC/1059, Q1/71, 23D10, 13E4, TFRC/1149, ER-MP21, YTA74.4, BU54, 2B6,RI7 217 (From US Patent App. 2011/0311544A1, filed Does not competeINSERM) Jun. 15, 2005, entitled “ANTI-CD71 with OKT9 BA120g MONOCLONALANTIBODIES AND USES THEREOF FOR TREATING MALIGNANT TUMOR CELLS” LUCA31U.S. Pat. No. 7,572,895, filed “LUCA31 epitope” Jun. 7, 2004, entitled“TRANSFERRIN RECEPTOR ANTIBODIES” (Salk Institute) Trowbridge, I. S. etal. “Anti-transferrin B3/25 receptor monoclonal antibody and T58/30toxin-antibody conjugates affect growth of human tumour cells.” Nature,1981, volume 294, pages 171-173 R17 217.1.3, Commercially availableanti-transferrin BioXcell 5E9C11, OKT9 receptor antibodies. 10Technology Dr., (BE0023 Suite 2B clone) West Lebanon, NH 03784-1671 USABK19.9, Gatter, K. C. et al. “Transferrin B3/25, T56/14 receptors inhuman tissues: their and T58/1 distribution and possible clinicalrelevance.” J Clin Pathol. 1983 May; 36(5): 539-45. Additional Anti-TfRSEQ ID NOs Anti-TfR antibody VH/VL CDR1 CDR2 CDR3 CDRH1 (SEQ ID NO:2265) VH1 2280 2273 2274 2267 CDRH2 (SEQ ID NO: 2266) VH2 2281 2273 22752267 CDRH3 (SEQ ID NO: 2267) VH3 2282 2273 2276 2267 CDRL1 (SEQ ID NO:2268) VH4 2283 2273 2275 2267 CDRL2 (SEQ ID NO: 2269) VL1 2284 2268 2269115 CDRL3 (SEQ ID NO: 2270) VL2 2285 2268 2269 115 VH (SEQ ID NO: 2271)VL3 2286 2268 2277 2270 VL (SEQ ID NO: 2272) VL4 2287 2278 2279 2270

In some embodiments, transferrin receptor antibodies of the presentdisclosure include one or more of the CDR-H (e.g., CDR-H1, CDR-H2, andCDR-H3) amino acid sequences from any one of the anti-transferrinreceptor antibodies selected from Table 8. In some embodiments,transferrin receptor antibodies include the CDR-H1, CDR-H2, and CDR-H3as provided for any one of the anti-transferrin receptor antibodiesselected from Table 8. In some embodiments, anti-transferrin receptorantibodies include the CDR-L1, CDR-L2, and CDR-L3 as provided for anyone of the anti-transferrin receptor antibodies selected from Table 8.In some embodiments, anti-transferrin antibodies include the CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one ofthe anti-transferrin receptor antibodies selected from Table 8. Thedisclosure also includes any nucleic acid sequence that encodes amolecule comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3as provided for any one of the anti-transferrin receptor antibodiesselected from Table 8. In some embodiments, antibody heavy and lightchain CDR3 domains may play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen. Accordingly,anti-transferrin receptor antibodies of the disclosure may include atleast the heavy and/or (e.g., and) light chain CDR3s of any one of theanti-transferrin receptor antibodies selected from Table 8.

In some examples, any of the anti-transferrin receptor antibodies of thedisclosure have one or more CDR (e.g., CDR-H or CDR-L) sequencessubstantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, and/or (e.g., and) CDR-L3 sequences from one of theanti-transferrin receptor antibodies selected from Table 8. In someembodiments, the position of one or more CDRs along the VH (e.g.,CDR-H1, CDR-H2, or CDR-H3) and/or (e.g., and) VL (e.g., CDR-L1, CDR-L2,or CDR-L3) region of an antibody described herein can vary by one, two,three, four, five, or six amino acid positions so long as immunospecificbinding to transferrin receptor (e.g., human transferrin receptor) ismaintained (e.g., substantially maintained, for example, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95% ofthe binding of the original antibody from which it is derived). Forexample, in some embodiments, the position defining a CDR of anyantibody described herein can vary by shifting the N-terminal and/or(e.g., and) C-terminal boundary of the CDR by one, two, three, four,five, or six amino acids, relative to the CDR position of any one of theantibodies described herein, so long as immunospecific binding totransferrin receptor (e.g., human transferrin receptor) is maintained(e.g., substantially maintained, for example, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% of thebinding of the original antibody from which it is derived). In anotherembodiment, the length of one or more CDRs along the VH (e.g., CDR-H1,CDR-H2, or CDR-H3) and/or (e.g., and) VL (e.g., CDR-L1, CDR-L2, orCDR-L3) region of an antibody described herein can vary (e.g., beshorter or longer) by one, two, three, four, five, or more amino acids,so long as immunospecific binding to transferrin receptor (e.g., humantransferrin receptor) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% of the binding of the original antibody fromwhich it is derived).

Accordingly, in some embodiments, a CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and/or (e.g., and) CDR-H3 described herein may be one, two,three, four, five or more amino acids shorter than one or more of theCDRs described herein (e.g., CDRS from any of the anti-transferrinreceptor antibodies selected from Table 8) so long as immunospecificbinding to transferrin receptor (e.g., human transferrin receptor) ismaintained (e.g., substantially maintained, for example, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%relative to the binding of the original antibody from which it isderived). In some embodiments, a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,and/or (e.g., and) CDR-H3 described herein may be one, two, three, four,five or more amino acids longer than one or more of the CDRs describedherein (e.g., CDRS from any of the anti-transferrin receptor antibodiesselected from Table 8) so long as immunospecific binding to transferrinreceptor (e.g., human transferrin receptor) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% relative to thebinding of the original antibody from which it is derived). In someembodiments, the amino portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and/or (e.g., and) CDR-H3 described herein can be extended byone, two, three, four, five or more amino acids compared to one or moreof the CDRs described herein (e.g., CDRS from any of theanti-transferrin receptor antibodies selected from Table 8) so long asimmunospecific binding to transferrin receptor (e.g., human transferrinreceptor is maintained (e.g., substantially maintained, for example, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% relative to the binding of the original antibody from which itis derived). In some embodiments, the carboxy portion of a CDR-L1,CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or (e.g., and) CDR-H3 describedherein can be extended by one, two, three, four, five or more aminoacids compared to one or more of the CDRs described herein (e.g., CDRSfrom any of the anti-transferrin receptor antibodies selected from Table8) so long as immunospecific binding to transferrin receptor (e.g.,human transferrin receptor) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% relative to the binding of theoriginal antibody from which it is derived). In some embodiments, theamino portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or (e.g.,and) CDR-H3 described herein can be shortened by one, two, three, four,five or more amino acids compared to one or more of the CDRs describedherein (e.g., CDRS from any of the anti-transferrin receptor antibodiesselected from Table 8) so long as immunospecific binding to transferrinreceptor (e.g., human transferrin receptor) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% relative to thebinding of the original antibody from which it is derived). In someembodiments, the carboxy portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and/or (e.g., and) CDR-H3 described herein can be shortened byone, two, three, four, five or more amino acids compared to one or moreof the CDRs described herein (e.g., CDRS from any of theanti-transferrin receptor antibodies selected from Table 8) so long asimmunospecific binding to transferrin receptor (e.g., human transferrinreceptor) is maintained (e.g., substantially maintained, for example, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% relative to the binding of the original antibody from which itis derived). Any method can be used to ascertain whether immunospecificbinding to transferrin receptor (e.g., human transferrin receptor) ismaintained, for example, using binding assays and conditions describedin the art.

In some examples, any of the anti-transferrin receptor antibodies of thedisclosure have one or more CDR (e.g., CDR-H or CDR-L) sequencessubstantially similar to any one of the anti-transferrin receptorantibodies selected from Table 8. For example, the antibodies mayinclude one or more CDR sequence(s) from any of the anti-transferrinreceptor antibodies selected from Table 8 containing up to 5, 4, 3, 2,or 1 amino acid residue variations as compared to the corresponding CDRregion in any one of the CDRs provided herein (e.g., CDRs from any ofthe anti-transferrin receptor antibodies selected from Table 8) so longas immunospecific binding to transferrin receptor (e.g., humantransferrin receptor) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% relative to the binding of the original antibodyfrom which it is derived). In some embodiments, any of the amino acidvariations in any of the CDRs provided herein may be conservativevariations. Conservative variations can be introduced into the CDRs atpositions where the residues are not likely to be involved ininteracting with a transferrin receptor protein (e.g., a humantransferrin receptor protein), for example, as determined based on acrystal structure. Some aspects of the disclosure provide transferrinreceptor antibodies that comprise one or more of the heavy chainvariable (VH) and/or (e.g., and) light chain variable (VL) domainsprovided herein. In some embodiments, any of the VH domains providedherein include one or more of the CDR-H sequences (e.g., CDR-H1, CDR-H2,and CDR-H3) provided herein, for example, any of the CDR-H sequencesprovided in any one of the anti-transferrin receptor antibodies selectedfrom Table 8. In some embodiments, any of the VL domains provided hereininclude one or more of the CDR-L sequences (e.g., CDR-L1, CDR-L2, andCDR-L3) provided herein, for example, any of the CDR-L sequencesprovided in any one of the anti-transferrin receptor antibodies selectedfrom Table 8.

In some embodiments, anti-transferrin receptor antibodies of thedisclosure include any antibody that includes a heavy chain variabledomain and/or (e.g., and) a light chain variable domain of anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 8. In someembodiments, anti-transferrin receptor antibodies of the disclosureinclude any antibody that includes the heavy chain variable and lightchain variable pairs of any anti-transferrin receptor antibody, such asany one of the anti-transferrin receptor antibodies selected from Table8.

Aspects of the disclosure provide anti-transferrin receptor antibodieshaving a heavy chain variable (VH) and/or (e.g., and) a light chainvariable (VL) domain amino acid sequence homologous to any of thosedescribed herein. In some embodiments, the anti-transferrin receptorantibody comprises a heavy chain variable sequence or a light chainvariable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%,or 99%) identical to the heavy chain variable sequence and/or any lightchain variable sequence of any anti-transferrin receptor antibody, suchas any one of the anti-transferrin receptor antibodies selected fromTable 8. In some embodiments, the homologous heavy chain variable and/or(e.g., and) a light chain variable amino acid sequences do not varywithin any of the CDR sequences provided herein. For example, in someembodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%,95%, 98%, or 99%) may occur within a heavy chain variable and/or (e.g.,and) a light chain variable sequence excluding any of the CDR sequencesprovided herein. In some embodiments, any of the anti-transferrinreceptor antibodies provided herein comprise a heavy chain variablesequence and a light chain variable sequence that comprises a frameworksequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identicalto the framework sequence of any anti-transferrin receptor antibody,such as any one of the anti-transferrin receptor antibodies selectedfrom Table 8.

In some embodiments, an anti-transferrin receptor antibody, whichspecifically binds to transferrin receptor (e.g., human transferrinreceptor), comprises a light chain variable VL domain comprising any ofthe CDR-L domains (CDR-L1, CDR-L2, and CDR-L3), or CDR-L domain variantsprovided herein, of any of the anti-transferrin receptor antibodiesselected from Table 8. In some embodiments, an anti-transferrin receptorantibody, which specifically binds to transferrin receptor (e.g., humantransferrin receptor), comprises a light chain variable VL domaincomprising the CDR-L1, the CDR-L2, and the CDR-L3 of anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 8. In someembodiments, the anti-transferrin receptor antibody comprises a lightchain variable (VL) region sequence comprising one, two, three or fourof the framework regions of the light chain variable region sequence ofany anti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 8. In someembodiments, the anti-transferrin receptor antibody comprises one, two,three or four of the framework regions of a light chain variable regionsequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical toone, two, three or four of the framework regions of the light chainvariable region sequence of any anti-transferrin receptor antibody, suchas any one of the anti-transferrin receptor antibodies selected fromTable 8. In some embodiments, the light chain variable framework regionthat is derived from said amino acid sequence consists of said aminoacid sequence but for the presence of up to 10 amino acid substitutions,deletions, and/or (e.g., and) insertions, preferably up to 10 amino acidsubstitutions. In some embodiments, the light chain variable frameworkregion that is derived from said amino acid sequence consists of saidamino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidresidues being substituted for an amino acid found in an analogousposition in a corresponding non-human, primate, or human light chainvariable framework region.

In some embodiments, an anti-transferrin receptor antibody thatspecifically binds to transferrin receptor comprises the CDR-L1, theCDR-L2, and the CDR-L3 of any anti-transferrin receptor antibody, suchas any one of the anti-transferrin receptor antibodies selected fromTable 8. In some embodiments, the antibody further comprises one, two,three or all four VL framework regions derived from the VL of a human orprimate antibody. The primate or human light chain framework region ofthe antibody selected for use with the light chain CDR sequencesdescribed herein, can have, for example, at least 70% (e.g., at least75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity with a lightchain framework region of a non-human parent antibody. The primate orhuman antibody selected can have the same or substantially the samenumber of amino acids in its light chain complementarity determiningregions to that of the light chain complementarity determining regionsof any of the antibodies provided herein, e.g., any of theanti-transferrin receptor antibodies selected from Table 8. In someembodiments, the primate or human light chain framework region aminoacid residues are from a natural primate or human antibody light chainframework region having at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity, atleast 98% identity, at least 99% (or more) identity with the light chainframework regions of any anti-transferrin receptor antibody, such as anyone of the anti-transferrin receptor antibodies selected from Table 8.In some embodiments, an anti-transferrin receptor antibody furthercomprises one, two, three or all four VL framework regions derived froma human light chain variable kappa subfamily. In some embodiments, ananti-transferrin receptor antibody further comprises one, two, three orall four VL framework regions derived from a human light chain variablelambda subfamily.

In some embodiments, any of the anti-transferrin receptor antibodiesprovided herein comprise a light chain variable domain that furthercomprises a light chain constant region. In some embodiments, the lightchain constant region is a kappa, or a lambda light chain constantregion. In some embodiments, the kappa or lambda light chain constantregion is from a mammal, e.g., from a human, monkey, rat, or mouse. Insome embodiments, the light chain constant region is a human kappa lightchain constant region. In some embodiments, the light chain constantregion is a human lambda light chain constant region. It should beappreciated that any of the light chain constant regions provided hereinmay be variants of any of the light chain constant regions providedherein. In some embodiments, the light chain constant region comprisesan amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or99% identical to any of the light chain constant regions of anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 8.

In some embodiments, the anti-transferrin receptor antibody is anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 8.

In some embodiments, an anti-transferrin receptor antibody comprises aVL domain comprising the amino acid sequence of any anti-transferrinreceptor antibody, such as any one of the anti-transferrin receptorantibodies selected from Table 8, and wherein the constant regionscomprise the amino acid sequences of the constant regions of an IgG,IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, or a human IgG, IgE,IgM, IgD, IgA or IgY immunoglobulin molecule. In some embodiments, ananti-transferrin receptor antibody comprises any of the VL domains, orVL domain variants, and any of the VH domains, or VH domain variants,wherein the VL and VH domains, or variants thereof, are from the sameantibody clone, and wherein the constant regions comprise the amino acidsequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgYimmunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulinmolecule. Non-limiting examples of human constant regions are describedin the art, e.g., see Kabat E A et al., (1991) supra.

In some embodiments, the muscle-targeting agent is a transferrinreceptor antibody (e.g., the antibody and variants thereof as describedin International Application Publication WO 2016/081643, incorporatedherein by reference).

The heavy chain and light chain CDRs of the antibody according todifferent definition systems are provided in Table 9. The differentdefinition systems, e.g., the Kabat definition, the Chothia definition,and/or (e.g., and) the contact definition have been described. See,e.g., (e.g., Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, Chothia et al., (1989)Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917,Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J.Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk andbioinf.org.uk/abs).

Table 9 Heavy chain and light chain CDRs of a mouse transferrin receptorantibody

TABLE 9 Heavy chain and light chain CDRs of amouse transferrin receptor antibody CDRs Kabat Chothia Contact CDR-H1SYWMH (SEQ GYTFTSY (SEQ TSYWMH (SEQ ID NO: 110) ID NO: 116) ID NO: 118)CDR-H2 EINPTNGRTNY NPTNGR (SEQ WIGEINPTNGR IEKFKS (SEQ ID NO: 117)TN (SEQ ID ID NO: 111) NO: 119) CDR-H3 GTRAYHY GTRAYHY (SEQ ARGTRA (SEQ(SEQ ID NO: ID NO: 112) ID NO: 120) 112) CDR-L1 RASDNLYSNLA RASDNLYSNLAYSNLAWY (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 113) 113) 121) CDR-L2DATNLAD DATNLAD (SEQ LLVYDATNLA (SEQ ID NO: ID NO: 114) (SEQ ID NO: 114)122) CDR-L3 QHFWGTPLT QHFWGTPLT QHFWGTPL (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 115) 115) 123)

The heavy chain variable domain (VH) and light chain variable domainsequences are also provided:

VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSS (SEQ IDNO: 124)

VL

VH (SEQ ID NO: 124) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGT RAYHYWGQGTSVT VL(SEQ ID NO: 125) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGA  GTKLELK

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the sameas the CDR-H1, CDR-H2, and CDR-H3 shown in Table 9. Alternatively or inaddition (e.g., in addition), the transferrin receptor antibody of thepresent disclosure comprises a CDR-L1, a CDR-L2, and a CDR-L3 that arethe same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 9.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3, whichcollectively contains no more than 5 amino acid variations (e.g., nomore than 5, 4, 3, 2, or 1 amino acid variation) as compared with theCDR-H1, CDR-H2, and CDR-H3 as shown in Table 9. “Collectively” meansthat the total number of amino acid variations in all of the three heavychain CDRs is within the defined range. Alternatively or in addition(e.g., in addition), the transferrin receptor antibody of the presentdisclosure may comprise a CDR-L1, a CDR-L2, and a CDR-L3, whichcollectively contains no more than 5 amino acid variations (e.g., nomore than 5, 4, 3, 2 or 1 amino acid variation) as compared with theCDR-L1, CDR-L2, and CDR-L3 as shown in Table 9.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3, at least one ofwhich contains no more than 3 amino acid variations (e.g., no more than3, 2, or 1 amino acid variation) as compared with the counterpart heavychain CDR as shown in Table 9. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosuremay comprise CDR-L1, a CDR-L2, and a CDR-L3, at least one of whichcontains no more than 3 amino acid variations (e.g., no more than 3, 2,or 1 amino acid variation) as compared with the counterpart light chainCDR as shown in Table 9.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-L3, which contains no more than 3 amino acidvariations (e.g., no more than 3, 2, or 1 amino acid variation) ascompared with the CDR-L3 as shown in Table 9. In some embodiments, thetransferrin receptor antibody of the present disclosure comprises aCDR-L3 containing one amino acid variation as compared with the CDR-L3as shown in Table 9. In some embodiments, the transferrin receptorantibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQID NO: 126) according to the Kabat and Chothia definition system) orQHFAGTPL (SEQ ID NO: 127) according to the Contact definition system).In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1 and a CDR-L2that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 9,and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) according to theKabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127)according to the Contact definition system).

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises heavy chain CDRs that collectively are at least 80%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs asshown in Table 9. Alternatively or in addition (e.g., in addition), thetransferrin receptor antibody of the present disclosure comprises lightchain CDRs that collectively are at least 80% (e.g., 80%, 85%, 90%, 95%,or 98%) identical to the light chain CDRs as shown in Table 9.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising the amino acid sequence of SEQ IDNO: 124. Alternatively or in addition (e.g., in addition), thetransferrin receptor antibody of the present disclosure comprises a VLcomprising the amino acid sequence of SEQ ID NO: 125.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VH as set forth in SEQ ID NO: 124.Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure comprises a VL containing nomore than 15 amino acid variations (e.g., no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VL as set forth in SEQ ID NO: 125.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as setforth in SEQ ID NO: 124. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosurecomprises a VL comprising an amino acid sequence that is at least 80%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth inSEQ ID NO: 125.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized antibody (e.g., a humanized variant of anantibody). In some embodiments, the transferrin receptor antibody of thepresent disclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, aCDR-L2, and a CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3shown in Table 9, and comprises a humanized heavy chain variable regionand/or (e.g., and) a humanized light chain variable region.

Humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some embodiments, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs derived from one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

In some embodiments, humanization is achieved by grafting the CDRs(e.g., as shown in Table 9) into the IGKV1-NL1*01 and IGHV1-3*01 humanvariable domains. In some embodiments, the transferrin receptor antibodyof the present disclosure is a humanized variant comprising one or moreamino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 ascompared with the VL as set forth in SEQ ID NO: 125, and/or (e.g., and)one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20,38, 40, 44, 66, 75, 81, 83, 87, and 108 as compared with the VH as setforth in SEQ ID NO: 124. In some embodiments, the transferrin receptorantibody of the present disclosure is a humanized variant comprisingamino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and70 as compared with the VL as set forth in SEQ ID NO: 125, and/or (e.g.,and) amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20,38, 40, 44, 66, 75, 81, 83, 87, and 108 as compared with the VH as setforth in SEQ ID NO: 124.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized antibody and contains the residues atpositions 43 and 48 of the VL as set forth in SEQ ID NO: 125.Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure is a humanized antibody andcontains the residues at positions 48, 67, 69, 71, and 73 of the VH asset forth in SEQ ID NO: 124.

The VH and VL amino acid sequences of an example humanized antibody thatmay be used in accordance with the present disclosure are provided:

Humanized VH (SEQ ID NO: 128)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCAR GTRAYHYWGQGTMVTVSSHumanized VL (SEQ ID NO: 129)DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTF GQGTKVEIK

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising the amino acid sequence of SEQ IDNO: 128. Alternatively or in addition (e.g., in addition), thetransferrin receptor antibody of the present disclosure comprises a VLcomprising the amino acid sequence of SEQ ID NO: 129.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VH as set forth in SEQ ID NO: 128.Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure comprises a VL containing nomore than 15 amino acid variations (e.g., no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VL as set forth in SEQ ID NO: 129.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as setforth in SEQ ID NO: 128. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosurecomprises a VL comprising an amino acid sequence that is at least 80%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth inSEQ ID NO: 129.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized variant comprising amino acid substitutions atone or more of positions 43 and 48 as compared with the VL as set forthin SEQ ID NO: 125, and/or (e.g., and) amino acid substitutions at one ormore of positions 48, 67, 69, 71, and 73 as compared with the VH as setforth in SEQ ID NO: 124. In some embodiments, the transferrin receptorantibody of the present disclosure is a humanized variant comprising aS43A and/or (e.g., and) a V48L mutation as compared with the VL as setforth in SEQ ID NO: 125, and/or (e.g., and) one or more of A67V, L69I,V71R, and K73T mutations as compared with the VH as set forth in SEQ IDNO: 124.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized variant comprising amino acid substitutions atone or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 ascompared with the VL as set forth in SEQ ID NO: 125, and/or (e.g., and)amino acid substitutions at one or more of positions 1, 5, 7, 11, 12,20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 ascompared with the VH as set forth in SEQ ID NO: 124.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a chimeric antibody, which can include a heavy constantregion and a light constant region from a human antibody. Chimericantibodies refer to antibodies having a variable region or part ofvariable region from a first species and a constant region from a secondspecies. Typically, in these chimeric antibodies, the variable region ofboth light and heavy chains mimics the variable regions of antibodiesderived from one species of mammals (e.g., a non-human mammal such asmouse, rabbit, and rat), while the constant portions are homologous tothe sequences in antibodies derived from another mammal such as human.In some embodiments, amino acid modifications can be made in thevariable region and/or (e.g., and) the constant region.

In some embodiments, the transferrin receptor antibody described hereinis a chimeric antibody, which can include a heavy constant region and alight constant region from a human antibody. Chimeric antibodies referto antibodies having a variable region or part of variable region from afirst species and a constant region from a second species. Typically, inthese chimeric antibodies, the variable region of both light and heavychains mimics the variable regions of antibodies derived from onespecies of mammals (e.g., a non-human mammal such as mouse, rabbit, andrat), while the constant portions are homologous to the sequences inantibodies derived from another mammal such as human. In someembodiments, amino acid modifications can be made in the variable regionand/or (e.g., and) the constant region.

In some embodiments, the heavy chain of any of the transferrin receptorantibodies as described herein may comprises a heavy chain constantregion (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combinationthereof). The heavy chain constant region can of any suitable origin,e.g., human, mouse, rat, or rabbit. In one specific example, the heavychain constant region is from a human IgG (a gamma heavy chain), e.g.,IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is givenbelow:

(SEQ ID NO: 130) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 

In some embodiments, the light chain of any of the transferrin receptorantibodies described herein may further comprise a light chain constantregion (CL), which can be any CL known in the art. In some examples, theCL is a kappa light chain. In other examples, the CL is a lambda lightchain. In some embodiments, the CL is a kappa light chain, the sequenceof which is provided below:

(SEQ ID NO: 83) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV  TKSFNRGEC

Other antibody heavy and light chain constant regions are well known inthe art, e.g., those provided in the IMGT database (www.imgt.org) or atwww.vbase2.org/vbstat.php., both of which are incorporated by referenceherein.

Examples of heavy chain and light chain amino acid sequences of thetransferrin receptor antibodies described are provided below:

Heavy Chain (VH + human IgG1 constant region) (SEQ ID NO: 132)QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK Light Chain (VL + kappa light chain) (SEQ ID NO: 133)DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHeavy Chain (humanized VH + human IgG1 constant region) (SEQ ID NO: 134)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKLight Chain (humanized VL + kappa light chain) (SEQ ID NO: 135)DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO:132. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprising anamino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or98%) identical to SEQ ID NO: 133. In some embodiments, the transferrinreceptor antibody described herein comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 132. Alternatively or in addition(e.g., in addition), the transferrin receptor antibody described hereincomprises a light chain comprising the amino acid sequence of SEQ ID NO:133.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in SEQ ID NO:132. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure comprises a light chaincontaining no more than 15 amino acid variations (e.g., no more than 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with the light chain as set forth in SEQ IDNO: 133.

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO:134. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprising anamino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or98%) identical to SEQ ID NO: 135. In some embodiments, the transferrinreceptor antibody described herein comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 134. Alternatively or in addition(e.g., in addition), the transferrin receptor antibody described hereincomprises a light chain comprising the amino acid sequence of SEQ ID NO:135.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain of humanized antibody as setforth in SEQ ID NO: 134. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosurecomprises a light chain containing no more than 15 amino acid variations(e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid variation) as compared with the light chainof humanized antibody as set forth in SEQ ID NO: 135.

In some embodiments, the transferrin receptor antibody is an antigenbinding fragment (Fab) of an intact antibody (full-length antibody).Antigen binding fragment of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fab′fragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments. Examples of Fab amino acid sequences of thetransferrin receptor antibodies described herein are provided below:

Heavy Chain Fab (VH + a portion of human IgG1 constant region)(SEQ ID NO: 136) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPHeavy Chain Fab (humanized VH + a portion of human IgG1 constant region)(SEQ ID NO: 137) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCP

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:136. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprisingthe amino acid sequence of SEQ ID NO: 133.

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:137. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprisingthe amino acid sequence of SEQ ID NO: 135.

The transferrin receptor antibodies described herein can be in anyantibody form, including, but not limited to, intact (i.e., full-length)antibodies, antigen-binding fragments thereof (such as Fab, Fab′,F(ab′)2, Fv), single chain antibodies, bi-specific antibodies, ornanobodies. In some embodiments, the transferrin receptor antibodydescribed herein is a scFv. In some embodiments, the transferrinreceptor antibody described herein is a scFv-Fab (e.g., scFv fused to aportion of a constant region). In some embodiments, the transferrinreceptor antibody described herein is a scFv fused to a constant region(e.g., human IgG1 constant region as set forth in SEQ ID NO: 130).

In some embodiments, any one of the anti-TfR antibodies described hereinis produced by recombinant DNA technology in Chinese hamster ovary (CHO)cell suspension culture, optionally in CHO-K1 cell (e.g., CHO-K1 cellsderived from European Collection of Animal Cell Culture, Cat. No.85051005) suspension culture.

In some embodiments, an antibody provided herein may have one or morepost-translational modifications. In some embodiments, N-terminalcyclization, also called pyroglutamate formation (pyro-Glu), may occurin the antibody at N-terminal Glutamate (Glu) and/or Glutamine (Gln)residues during production. As such, it should be appreciated that anantibody specified as having a sequence comprising an N-terminalglutamate or glutamine residue encompasses antibodies that haveundergone pyroglutamate formation resulting from a post-translationalmodification. In some embodiments, pyroglutamate formation occurs in aheavy chain sequence. In some embodiments, pyroglutamate formationoccurs in a light chain sequence.

b. Other Muscle-Targeting Antibodies

In some embodiments, the muscle-targeting antibody is an antibody thatspecifically binds hemojuvelin, caveolin-3, Duchenne muscular dystrophypeptide, myosin Iib, or CD63. In some embodiments, the muscle-targetingantibody is an antibody that specifically binds a myogenic precursorprotein. Exemplary myogenic precursor proteins include, withoutlimitation, ABCG2, M-Cadherin/Cadherin-15, Caveolin-1, CD34, FoxK1,Integrin alpha 7, Integrin alpha 7 beta 1, MYF-5, MyoD, Myogenin,NCAM-1/CD56, Pax3, Pax7, and Pax9. In some embodiments, themuscle-targeting antibody is an antibody that specifically binds askeletal muscle protein. Exemplary skeletal muscle proteins include,without limitation, alpha-Sarcoglycan, beta-Sarcoglycan, CalpainInhibitors, Creatine Kinase MM/CKMM, eIF5A, Enolase 2/Neuron-specificEnolase, epsilon-Sarcoglycan, FABP3/H-FABP, GDF-8/Myostatin,GDF-11/GDF-8, Integrin alpha 7, Integrin alpha 7 beta 1, Integrin beta1/CD29, MCAM/CD146, MyoD, Myogenin, Myosin Light Chain KinaseInhibitors, NCAM-1/CD56, and Troponin I. In some embodiments, themuscle-targeting antibody is an antibody that specifically binds asmooth muscle protein. Exemplary smooth muscle proteins include, withoutlimitation, alpha-Smooth Muscle Actin, VE-Cadherin, Caldesmon/CALD1,Calponin 1, Desmin, Histamine H2 R, Motilin R/GPR38, Transgelin/TAGLN,and Vimentin. However, it should be appreciated that antibodies toadditional targets are within the scope of this disclosure and theexemplary lists of targets provided herein are not meant to be limiting.

c. Antibody Features/Alterations

In some embodiments, conservative mutations can be introduced intoantibody sequences (e.g., CDRs or framework sequences) at positionswhere the residues are not likely to be involved in interacting with atarget antigen (e.g., transferrin receptor), for example, as determinedbased on a crystal structure. In some embodiments, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of a muscle-targeting antibody described herein (e.g., in a CH2domain (residues 231-340 of human IgG1) and/or (e.g., and) CH3 domain(residues 341-447 of human IgG1) and/or (e.g., and) the hinge region,with numbering according to the Kabat numbering system (e.g., the EUindex in Kabat)) to alter one or more functional properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding and/or (e.g., and) antigen-dependent cellular cytotoxicity.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain can be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of a muscle-targetingantibody described herein (e.g., in a CH2 domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) CH3 domain (residues 341-447 of humanIgG1) and/or (e.g., and) the hinge region, with numbering according tothe Kabat numbering system (e.g., the EU index in Kabat)) to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to decrease the half-life of theanti-transferrin receptor antibody in vivo. In some embodiments, one,two or more amino acid mutations (i.e., substitutions, insertions ordeletions) are introduced into an IgG constant domain, or FcRn-bindingfragment thereof (preferably an Fc or hinge-Fc domain fragment) toincrease the half-life of the antibody in vivo. In some embodiments, theantibodies can have one or more amino acid mutations (e.g.,substitutions) in the second constant (CH2) domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) the third constant (CH3) domain (residues341-447 of human IgG1), with numbering according to the EU index inKabat (Kabat E A et al., (1991) supra). In some embodiments, theconstant region of the IgG1 of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU index as in Kabat. See U.S. Pat. No. 7,658,921, which isincorporated herein by reference. This type of mutant IgG, referred toas “YTE mutant” has been shown to display fourfold increased half-lifeas compared to wild-type versions of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, anantibody comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU index as in Kabat.

In some embodiments, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the anti-transferrin receptor antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments,the deletion or inactivation (through point mutations or other means) ofa constant region domain can reduce Fc receptor binding of thecirculating antibody thereby increasing tumor localization. See, e.g.,U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description of mutationsthat delete or inactivate the constant domain and thereby increase tumorlocalization. In some embodiments, one or more amino acid substitutionsmay be introduced into the Fc region of an antibody described herein toremove potential glycosylation sites on Fc region, which may reduce Fcreceptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino in the constant region of amuscle-targeting antibody described herein can be replaced with adifferent amino acid residue such that the antibody has altered Clqbinding and/or (e.g., and) reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al). In some embodiments, one or moreamino acid residues in the N-terminal region of the CH2 domain of anantibody described herein are altered to thereby alter the ability ofthe antibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments, the Fcregion of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or (e.g., and) to increase the affinity of theantibody for an Fcγ receptor. This approach is described further inInternational Publication No. WO 00/42072.

In some embodiments, the heavy and/or (e.g., and) light chain variabledomain(s) sequence(s) of the antibodies provided herein can be used togenerate, for example, CDR-grafted, chimeric, humanized, or compositehuman antibodies or antigen-binding fragments, as described elsewhereherein. As understood by one of ordinary skill in the art, any variant,CDR-grafted, chimeric, humanized, or composite antibodies derived fromany of the antibodies provided herein may be useful in the compositionsand methods described herein and will maintain the ability tospecifically bind transferrin receptor, such that the variant,CDR-grafted, chimeric, humanized, or composite antibody has at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% or more binding to transferrin receptor relative to the originalantibody from which it is derived.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like hinge sequence. Accordingly, any of theantibodies may include a stabilizing ‘Adair’ mutation.

As provided herein, antibodies of this disclosure may optionallycomprise constant regions or parts thereof. For example, a VL domain maybe attached at its C-terminal end to a light chain constant domain likeCκ or Cλ. Similarly, a VH domain or portion thereof may be attached toall or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and anyisotype subclass. Antibodies may include suitable constant regions (see,for example, Kabat et al., Sequences of Proteins of ImmunologicalInterest, No. 91-3242, National Institutes of Health Publications,Bethesda, Md. (1991)). Therefore, antibodies within the scope of thismay disclosure include VH and VL domains, or an antigen binding portionthereof, combined with any suitable constant regions.

ii. Muscle-Targeting Peptides

Some aspects of the disclosure provide muscle-targeting peptides asmuscle-targeting agents. Short peptide sequences (e.g., peptidesequences of 5-20 amino acids in length) that bind to specific celltypes have been described. For example, cell-targeting peptides havebeen described in Vines e., et al., A. “Cell-penetrating andcell-targeting peptides in drug delivery” Biochim Biophys Acta 2008,1786: 126-38; Jarver P., et al., “In vivo biodistribution and efficacyof peptide mediated delivery” Trends Pharmacol Sci 2010; 31: 528-35;Samoylova T. I., et al., “Elucidation of muscle-binding peptides byphage display screening” Muscle Nerve 1999; 22: 460-6; U.S. Pat. No.6,329,501, issued on Dec. 11, 2001, entitled “METHODS AND COMPOSITIONSFOR TARGETING COMPOUNDS TO MUSCLE”; and Samoylov A. M., et al.,“Recognition of cell-specific binding of phage display derived peptidesusing an acoustic wave sensor.” Biomol Eng 2002; 18: 269-72; the entirecontents of each of which are incorporated herein by reference. Bydesigning peptides to interact with specific cell surface antigens(e.g., receptors), selectivity for a desired tissue, e.g., muscle, canbe achieved. Skeletal muscle-targeting has been investigated and a rangeof molecular payloads are able to be delivered. These approaches mayhave high selectivity for muscle tissue without many of the practicaldisadvantages of a large antibody or viral particle. Accordingly, insome embodiments, the muscle-targeting agent is a muscle-targetingpeptide that is from 4 to 50 amino acids in length. In some embodiments,the muscle-targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 amino acids in length. Muscle-targeting peptides can be generatedusing any of several methods, such as phage display.

In some embodiments, a muscle-targeting peptide may bind to aninternalizing cell surface receptor that is overexpressed or relativelyhighly expressed in muscle cells, e.g. a transferrin receptor, comparedwith certain other cells. In some embodiments, a muscle-targetingpeptide may target, e.g., bind to, a transferrin receptor. In someembodiments, a peptide that targets a transferrin receptor may comprisea segment of a naturally occurring ligand, e.g., transferrin. In someembodiments, a peptide that targets a transferrin receptor is asdescribed in U.S. Pat. No. 6,743,893, filed Nov. 30, 2000,“RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRINRECEPTOR”. In some embodiments, a peptide that targets a transferrinreceptor is as described in Kawamoto, M. et al, “A novel transferrinreceptor-targeted hybrid peptide disintegrates cancer cell membrane toinduce rapid killing of cancer cells.” BMC Cancer. 2011 Aug. 18; 11:359.In some embodiments, a peptide that targets a transferrin receptor is asdescribed in U.S. Pat. No. 8,399,653, filed May 20, 2011,“TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY”.

As discussed above, examples of muscle targeting peptides have beenreported. For example, muscle-specific peptides were identified usingphage display library presenting surface heptapeptides. As one example apeptide having the amino acid sequence ASSLNIA (SEQ ID NO: 138) bound toC2C12 murine myotubes in vitro, and bound to mouse muscle tissue invivo. Accordingly, in some embodiments, the muscle-targeting agentcomprises the amino acid sequence ASSLNIA (SEQ ID NO: 138). This peptidedisplayed improved specificity for binding to heart and skeletal muscletissue after intravenous injection in mice with reduced binding toliver, kidney, and brain. Additional muscle-specific peptides have beenidentified using phage display. For example, a 12 amino acid peptide wasidentified by phage display library for muscle targeting in the contextof treatment for DMD. See, Yoshida D., et al., “Targeting of salicylateto skin and muscle following topical injections in rats.” Int J Pharm2002; 231: 177-84; the entire contents of which are hereby incorporatedby reference. Here, a 12 amino acid peptide having the sequenceSKTFNTHPQSTP (SEQ ID NO: 139) was identified and this muscle-targetingpeptide showed improved binding to C2C12 cells relative to the ASSLNIA(SEQ ID NO: 138) peptide.

An additional method for identifying peptides selective for muscle(e.g., skeletal muscle) over other cell types includes in vitroselection, which has been described in Ghosh D., et al., “Selection ofmuscle-binding peptides from context-specific peptide-presenting phagelibraries for adenoviral vector targeting” J Virol 2005; 79: 13667-72;the entire contents of which are incorporated herein by reference. Bypre-incubating a random 12-mer peptide phage display library with amixture of non-muscle cell types, non-specific cell binders wereselected out. Following rounds of selection the 12 amino acid peptideTARGEHKEEELI (SEQ ID NO: 140) appeared most frequently. Accordingly, insome embodiments, the muscle-targeting agent comprises the amino acidsequence TARGEHKEEELI (SEQ ID NO: 140).

A muscle-targeting agent may an amino acid-containing molecule orpeptide. A muscle-targeting peptide may correspond to a sequence of aprotein that preferentially binds to a protein receptor found in musclecells. In some embodiments, a muscle-targeting peptide contains a highpropensity of hydrophobic amino acids, e.g. valine, such that thepeptide preferentially targets muscle cells. In some embodiments, amuscle-targeting peptide has not been previously characterized ordisclosed. These peptides may be conceived of, produced, synthesized,and/or (e.g., and) derivatized using any of several methodologies, e.g.phage displayed peptide libraries, one-bead one-compound peptidelibraries, or positional scanning synthetic peptide combinatoriallibraries. Exemplary methodologies have been characterized in the artand are incorporated by reference (Gray, B. P. and Brown, K. C.“Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” ChemRev. 2014, 114:2, 1020-1081.; Samoylova, T. I. and Smith, B. F.“Elucidation of muscle-binding peptides by phage display screening.”Muscle Nerve, 1999, 22:4. 460-6.). In some embodiments, amuscle-targeting peptide has been previously disclosed (see, e.g. WriterM. J. et al. “Targeted gene delivery to human airway epithelial cellswith synthetic vectors incorporating novel targeting peptides selectedby phage display.” J. Drug Targeting. 2004; 12:185; Cal, D.“BDNF-mediated enhancement of inflammation and injury in the agingheart.” Physiol Genomics. 2006, 24:3, 191-7.; Zhang, L. “Molecularprofiling of heart endothelial cells.” Circulation, 2005, 112:11,1601-11.; McGuire, M. J. et al. “In vitro selection of a peptide withhigh selectivity for cardiomyocytes in vivo.” J Mol Biol. 2004, 342:1,171-82.). Exemplary muscle-targeting peptides comprise an amino acidsequence of the following group: CQAQGQLVC (SEQ ID NO: 141), CSERSMNFC(SEQ ID NO: 142), CPKTRRVPC (SEQ ID NO: 143), WLSEAGPVVTVRALRGTGSW (SEQID NO: 144), ASSLNIA (SEQ ID NO: 138), CMQHSMRVC (SEQ ID NO: 145), andDDTRHWG (SEQ ID NO: 146). In some embodiments, a muscle-targetingpeptide may comprise about 2-25 amino acids, about 2-20 amino acids,about 2-15 amino acids, about 2-10 amino acids, or about 2-5 aminoacids. Muscle-targeting peptides may comprise naturally-occurring aminoacids, e.g. cysteine, alanine, or non-naturally-occurring or modifiedamino acids. Non-naturally occurring amino acids include β-amino acids,homo-amino acids, proline derivatives, 3-substituted alaninederivatives, linear core amino acids, N-methyl amino acids, and othersknown in the art. In some embodiments, a muscle-targeting peptide may belinear; in other embodiments, a muscle-targeting peptide may be cyclic,e.g. bicyclic (see, e.g. Silvana, M. G. et al. Mol. Therapy, 2018, 26:1,132-147.).

iii. Muscle-Targeting Receptor Ligands

A muscle-targeting agent may be a ligand, e.g. a ligand that binds to areceptor protein. A muscle-targeting ligand may be a protein, e.g.transferrin, which binds to an internalizing cell surface receptorexpressed by a muscle cell. Accordingly, in some embodiments, themuscle-targeting agent is transferrin, or a derivative thereof thatbinds to a transferrin receptor. A muscle-targeting ligand mayalternatively be a small molecule, e.g. a lipophilic small molecule thatpreferentially targets muscle cells relative to other cell types.Exemplary lipophilic small molecules that may target muscle cellsinclude compounds comprising cholesterol, cholesteryl, stearic acid,palmitic acid, oleic acid, oleyl, linolene, linoleic acid, myristicacid, sterols, dihydrotestosterone, testosterone derivatives, glycerine,alkyl chains, trityl groups, and alkoxy acids.

iv. Muscle-Targeting Aptamers

A muscle-targeting agent may be an aptamer, e.g. an RNA aptamer, whichpreferentially targets muscle cells relative to other cell types. Insome embodiments, a muscle-targeting aptamer has not been previouslycharacterized or disclosed. These aptamers may be conceived of,produced, synthesized, and/or (e.g., and) derivatized using any ofseveral methodologies, e.g. Systematic Evolution of Ligands byExponential Enrichment. Exemplary methodologies have been characterizedin the art and are incorporated by reference (Yan, A. C. and Levy, M.“Aptamers and aptamer targeted delivery” RNA biology, 2009, 6:3,316-20.; Germer, K. et al. “RNA aptamers and their therapeutic anddiagnostic applications.” Int. J. Biochem. Mol. Biol. 2013; 4: 27-40.).In some embodiments, a muscle-targeting aptamer has been previouslydisclosed (see, e.g. Phillippou, S. et al. “Selection and Identificationof Skeletal-Muscle-Targeted RNA Aptamers.” Mol Ther Nucleic Acids. 2018,10:199-214.; Thiel, W. H. et al. “Smooth Muscle Cell-targeted RNAAptamer Inhibits Neointimal Formation.” Mol Ther. 2016, 24:4, 779-87.).Exemplary muscle-targeting aptamers include the A01B RNA aptamer and RNAApt 14. In some embodiments, an aptamer is a nucleic acid-based aptamer,an oligonucleotide aptamer or a peptide aptamer. In some embodiments, anaptamer may be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about1-5 Da, about 1-3 kDa, or smaller.

v. Other Muscle-Targeting Agents

One strategy for targeting a muscle cell (e.g., a skeletal muscle cell)is to use a substrate of a muscle transporter protein, such as atransporter protein expressed on the sarcolemma. In some embodiments,the muscle-targeting agent is a substrate of an influx transporter thatis specific to muscle tissue. In some embodiments, the influxtransporter is specific to skeletal muscle tissue. Two main classes oftransporters are expressed on the skeletal muscle sarcolemma, (1) theadenosine triphosphate (ATP) binding cassette (ABC) superfamily, whichfacilitate efflux from skeletal muscle tissue and (2) the solute carrier(SLC) superfamily, which can facilitate the influx of substrates intoskeletal muscle. In some embodiments, the muscle-targeting agent is asubstrate that binds to an ABC superfamily or an SLC superfamily oftransporters. In some embodiments, the substrate that binds to the ABCor SLC superfamily of transporters is a naturally-occurring substrate.In some embodiments, the substrate that binds to the ABC or SLCsuperfamily of transporters is a non-naturally occurring substrate, forexample, a synthetic derivative thereof that binds to the ABC or SLCsuperfamily of transporters.

In some embodiments, the muscle-targeting agent is a substrate of an SLCsuperfamily of transporters. SLC transporters are either equilibrativeor use proton or sodium ion gradients created across the membrane todrive transport of substrates. Exemplary SLC transporters that have highskeletal muscle expression include, without limitation, the SATTtransporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-Jtransporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2transporter (FLJ46769; SLC22A5), ENT transporters (ENT1; SLC29A1 andENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter(KIAA1382; SLC38A2). These transporters can facilitate the influx ofsubstrates into skeletal muscle, providing opportunities for muscletargeting.

In some embodiments, the muscle-targeting agent is a substrate of anequilibrative nucleoside transporter 2 (ENT2) transporter. Relative toother transporters, ENT2 has one of the highest mRNA expressions inskeletal muscle. While human ENT2 (hENT2) is expressed in most bodyorgans such as brain, heart, placenta, thymus, pancreas, prostate, andkidney, it is especially abundant in skeletal muscle. Human ENT2facilitates the uptake of its substrates depending on theirconcentration gradient. ENT2 plays a role in maintaining nucleosidehomeostasis by transporting a wide range of purine and pyrimidinenucleobases. The hENT2 transporter has a low affinity for allnucleosides (adenosine, guanosine, uridine, thymidine, and cytidine)except for inosine. Accordingly, in some embodiments, themuscle-targeting agent is an ENT2 substrate. Exemplary ENT2 substratesinclude, without limitation, inosine, 2′,3′-dideoxyinosine, andcalofarabine. In some embodiments, any of the muscle-targeting agentsprovided herein are associated with a molecular payload (e.g.,oligonucleotide payload). In some embodiments, the muscle-targetingagent is covalently linked to the molecular payload. In someembodiments, the muscle-targeting agent is non-covalently linked to themolecular payload.

In some embodiments, the muscle-targeting agent is a substrate of anorganic cation/carnitine transporter (OCTN2), which is a sodiumion-dependent, high affinity carnitine transporter. In some embodiments,the muscle-targeting agent is carnitine, mildronate, acetylcarnitine, orany derivative thereof that binds to OCTN2. In some embodiments, thecarnitine, mildronate, acetylcarnitine, or derivative thereof iscovalently linked to the molecular payload (e.g., oligonucleotidepayload).

A muscle-targeting agent may be a protein that is protein that exists inat least one soluble form that targets muscle cells. In someembodiments, a muscle-targeting protein may be hemojuvelin (also knownas repulsive guidance molecule C or hemochromatosis type 2 protein), aprotein involved in iron overload and homeostasis. In some embodiments,hemojuvelin may be full length or a fragment, or a mutant with at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98% or at least 99% sequence identity to a functional hemojuvelinprotein. In some embodiments, a hemojuvelin mutant may be a solublefragment, may lack a N-terminal signaling, and/or (e.g., and) lack aC-terminal anchoring domain. In some embodiments, hemojuvelin may beannotated under GenBank RefSeq Accession Numbers NM_001316767.1,NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should beappreciated that a hemojuvelin may be of human, non-human primate, orrodent origin.

B. Molecular Payloads

Some aspects of the disclosure provide molecular payloads, e.g., formodulating a biological outcome, e.g., the transcription of a DNAsequence, the splicing and processing of a RNA sequence, the expressionof a protein, or the activity of a protein. In some embodiments, amolecular payload is linked to, or otherwise associated with amuscle-targeting agent. In some embodiments, such molecular payloads arecapable of targeting to a muscle cell, e.g., via specifically binding toa nucleic acid or protein in the muscle cell following delivery to themuscle cell by an associated muscle-targeting agent. It should beappreciated that various types of muscle-targeting agents may be used inaccordance with the disclosure. For example, the molecular payload maycomprise, or consist of, an oligonucleotide (e.g., antisenseoligonucleotide), a peptide (e.g., a peptide that binds a nucleic acidor protein associated with disease in a muscle cell), a protein (e.g., aprotein that binds a nucleic acid or protein associated with disease ina muscle cell), or a small molecule (e.g., a small molecule thatmodulates the function of a nucleic acid or protein associated withdisease in a muscle cell). In some embodiments, the molecular payload isan oligonucleotide that comprises a strand having a region ofcomplementarity to a mutated DMD allele. Exemplary molecular payloadsare described in further detail herein, however, it should beappreciated that the exemplary molecular payloads provided herein arenot meant to be limiting.

i. Oligonucleotides

Any suitable oligonucleotide may be used as a molecular payload, asdescribed herein. In some embodiments, the oligonucleotide may bedesigned to induce exon skipping, e.g., EXONDYS 51 oligonucleotide(Sarepta Therapeutics, Inc.), which comprises SEQ ID NO: 343(CUCCAACAUCAAGGAAGAUGGCAUUUCUAG); WVE-210201 (Wave Life Sciences), whichcomprises SEQ ID NO: 334 (UCAAGGAAGAUGGCAUUUCU); Casimersen (SareptaTherapeutics, Inc.), which comprises SEQ ID NO: 302(CAAUGCCAUCCUGGAGUUCCUG); or Golodirsen (Sarepta Therapeutics, Inc.),which comprises SEQ ID NO: 380 (GUUGCCUCCGGUUCUGAAGGUGUUC). In someembodiments, the oligonucleotide may be designed to induce exonskipping, e.g., viltolarsen (NS Pharma, Inc.), which comprises SEQ IDNO: 2257 (CCTCCGGTTCTGAAGGTGTTC) or renadirsen (Daiichi Sankyo Company),which comprises SEQ ID NO: 2252 (CGCUGCCCAAUGCCAUCC). In someembodiments, the oligonucleotide comprises a sequence or portion thereof(e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutivenucleosides thereof) of a sequence provided in Table 10, and/or theoligonucleotide comprises a region of complementarity to a targetsequence provided in Table 10. Any one or more of the thymine bases(T's) in any one of the oligonucleotides provided herein (e.g., theoligonucleotides listed in Table 10) may optionally be uracil bases(U's), and/or any one or more of the U's in the oligonucleotidesprovided herein may optionally be T's.

TABLE 10 Examples of oligonucleotide molecular payloads SEQ SEQ SEQ IDAntisense ID Antisense ID Target Name NO: Sequence^(†) NO: Sequence^(†)NO: Sequence^(†) EXONDYS 51  343 CUCCAACAUC  745 CTCCAACATC 1550CTAGAAATGC AAGGAAGAUG AAGGAAGATG CATCTTCCTTG GCAUUUCUAG GCATTTCTAGATGTTGGAG WVE-210201  334 UCAAGGAAGA 2254 TCAAGGAAGA 2259 AGAAATGCCAUGGCAUUUCU TGGCATTTCT TCTTCCTTGA Casimersen  302 CAAUGCCAUC 2255CAATGCCATC 2260 CAGGAACTCC CUGGAGUUCC CTGGAGTTCC AGGATGGCAT UG TG TGGolodirsen  380 GUUGCCUCCG 2256 GTTGCCTCCG 2261 GAACACCTTC GUUCUGAAGGGTTCTGAAGG AGAACCGGAG UGUUC TGTTC GCAAC Viltolarsen 2251 CCUCCGGUUC 2257CCTCCGGTTCT 2262 GAACACCTTC UGAAGGUGUU GAAGGTGTTC AGAACCGGAG C GRenadirsen 2252 CGCUGCCCAA 2258 CGCTGCCCAA 2263 GGATGGCATT UGCCAUCCTGCCATCC GGGCAGCG ^(†)Each thymine base (T) in any one of theoligonucleotides and/or target sequences provided in Table 10 mayindependently and optionally be replaced with a uracil base (U), and/oreach U may independently and optionally be replaced with a T. Targetsequences listed in Table 10 contain Ts, but binding of a DMD-targetingoligonucleotide to RNA and/or DNA is contemplated.

In some embodiments, the oligonucleotide may be designed to causedegradation of an mRNA (e.g., the oligonucleotide may be a gapmer, ansiRNA, a ribozyme or an aptamer that causes degradation). In someembodiments, the oligonucleotide may be designed to block translation ofan mRNA (e.g., the oligonucleotide may be a mixmer, an siRNA or anaptamer that blocks translation). In some embodiments, anoligonucleotide may be designed to cause degradation and blocktranslation of an mRNA. In some embodiments, the oligonucleotide may bedesigned to promote stability of an mRNA. In some embodiments, theoligonucleotide may be designed to promote translation of an mRNA. Insome embodiments, an oligonucleotide may be designed to promotestability and promote translation of an mRNA. In some embodiments, anoligonucleotide may be a guide nucleic acid (e.g., guide RNA) fordirecting activity of an enzyme (e.g., a gene editing enzyme). In someembodiments, a guide nucleic acid may direct an enzyme to delete theentirety or a part of a mutated DMD allele (e.g., to facilitate in-frameexon skipping). In some embodiments, the oligonucleotide may be designedto target repressive regulators of DMD expression, e.g., miR-31. Otherexamples of oligonucleotides are provided herein. It should beappreciated that, in some embodiments, oligonucleotides in one format(e.g., antisense oligonucleotides) may be suitably adapted to anotherformat (e.g., siRNA oligonucleotides) by incorporating functionalsequences (e.g., antisense strand sequences) from one format to theother format.

Examples of oligonucleotides useful for targeting DMD are provided inU.S. Patent Application Publication US20100130591A1, published on May27, 2010, entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD”; U.S.Pat. No. 8,361,979, issued Jan. 29, 2013, entitled “MEANS AND METHOD FORINDUCING EXON-SKIPPING”; U.S. Patent Application Publication20120059042, published Mar. 8, 2012, entitled “METHOD FOR EFFICIENT EXON(44) SKIPPING IN DUCHENNE MUSCULAR DYSTROPHY AND ASSOCIATED MEANS; U.S.Patent Application Publication 20140329881, published Nov. 6, 2014,entitled “EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY”;U.S. Pat. No. 8,232,384, issued Jul. 31, 2012, entitled “ANTISENSEOLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF”;U.S. Patent Application Publication 20120022134A1, published Jan. 26,2012, entitled “METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 INDUCHENNE MUSCULAR DYSTROPHY PRE-MRNA; U.S. Patent ApplicationPublication 20120077860, published Mar. 29, 2012, entitled“ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING ADISPENSABLE DOMAN PROTEIN”; U.S. Pat. No. 8,324,371, issued Dec. 4,2012, entitled “OLIGOMERS”; U.S. Pat. No. 9,078,911, issued Jul. 14,2015, entitled “ANTISENSE OLIGONUCLEOTIDES”; U.S. Pat. No. 9,079,934,issued Jul. 14, 2015, entitled “ANTISENSE NUCLEIC ACIDS”; U.S. Pat. No.9,034,838, issued May 19, 2015, entitled “MIR-31 IN DUCHENNE MUSCULARDYSTROPHY THERAPY”; and International Patent Publication WO2017062862A3,published Apr. 13, 2017, entitled “OLIGONUCLEOTIDE COMPOSITIONS ANDMETHODS THEREOF”; the contents of each of which are incorporated hereinin their entireties.

Table 14 provides non-limiting examples of sequences of oligonucleotidesthat are useful for targeting DMD, e.g., for exon skipping. In someembodiments, an oligonucleotide may comprise any sequence provided inTable 14.

TABLE 14 Oligonucleotide sequences for targeting DMD. SEQ ID EXON NO:SEQUENCE  8 151 CUUCCUGGAUGGCUUCAAU  8 152 GUACAUUAAGAUGGACUUC  8 153UAUCUGGAUAGGUGGUAUCAAGAUCUGUAA  8 154 AUGUAACUGAAAAUGUUCUUCUUUA  8 155UGGAUAGGUGGUAUCAACAUCUGUAAGCAC  8 156 GAUAGGUGGUAUCAACAUCUGU  8 157UAUCUGGAUAGGUGGUAUCAAGAUCUGUAA  8 158 AAACUUGGAAGAGUGAUGUGAUGUA  8 159GCUCACUUGUUGAGGCAAAACUUGGAA  8 160 GCCUUGGCAACAUUUCCACUUCCUG  8 161UACACACUUUACCUGUUGAGAAUAG  8 162 GAUAGGUGGUAUCAACAUCUGUAA  8 163GAUAGGUGGUAUCAACAUCUG  8 164 GAUAGGUGGUAUCAACAUCUGUAAG  8 165GGUGGUAUCAACAUCUGUAA  8 166 GUAUCAACAUCUGUAAGCAC 23 167CGGCUAAUUUCAGAGGGCGCUUUCUUNGAC 23 168 ACAGUGGUGCUGAGAUAGUAUAGGCC 23 169UAGGCCACUUUGUUGCUCUUGC 23 170 UUCAGAGGGCGCUUUCUUC 23 171GGCCAAACCUCGGCUUACCUGAAAU 23 172 GGCCAAACCUCGGCUUACCU 35 173UCUUCAGGUGCACCUUCUGUUUCUCAAUCU 35 174 UCUGUGAUACUCUUCAGGUGCACCUUCUGU 35175 UCUUCUGCUCGGGAGGUGACA 35 176 CCAGUUACUAUUCAGAAGAC 35 177UCUUCAGGUGCACCUUCUGU 43 178 UGCUGCUGUCUUCUUGCU 43 179UUGUUAACUUUUUCCCAUU 43 180 UGUUAACUUUUUCCCAUUGG 43 181CAUUUUGUUAACUUUUUCCC 43 182 CUGUAGCUUCACCCUUUCC 43 183GAGAGCUUCCUGUAGCUUCACCCUUU 43 184 UCCUGUAGCUUCACCCUUUCCACAGGCG 43 185UGUGUUACCUACCCUUGUCG 43 186 UAGACUAUCUUUUAUAUUCUGUAAUAU 43 187GAGAGCUUCCUGUAGCUUCACCCUUUCCA 43 188 UUCCUGUAGCUUCACCCUUUCCACAGGCGUU 43189 AGCUUCCUGUAGCUUCACCCUUU 43 190 GGAGAGAGCUUCCUGUAGCUUCACCCUUU 43 191GAGAGCUUCCUGUAGCUUCACCC 43 192 UAUGUGUUACCUACCCUUGUCGGUC 43 193GGAGAGAGCUUCCUGUAGCU 43 194 UCACCCUUUCCACAGGCGUUGCA 43 195GCUGGGAGAGAGCUUCCUGUAGCUUCAC 43 196 UGUUACCUACCCUUGUCGGUCCUUGUAC 43 197CUGCUGUCUUCUUGCUAUGAAUAAUGUC 43 198 GGCGUUGCACUUUGCAAUGCUGCUGUCU 43 199UUGGAAAUCAAGCUGGGAGAGAGCUUCC 43 200 CUACCCUUGUCGGUCCUUGUACAUUUUG 43 201GUCAAUCCGACCUGAGCUUUGUUGUAGA 43 202 CUUGCUAUGAAUAAUGUCAAUCCGACC 43 203UAUAUGUGUUACCUACCCUUGUCGGUCC 43 204 AAUCAGCUGGGAGAGAGCUUCCUGUAGCU 43 205UCGUUCUUCUGUCGUCGUAACGUUUC 44 206 UUUGUGUCUUUCUGAGAAAC 44 207AAAGACUUACCUUAAGAUAC 44 208 AUCUGUCAAAUCGCCUGCAG 44 209CGCCGCCAUUUCUCAACAG 44 210 UUUGUAUUUAGCAUGUUCCC 44 211 CCGCCAUUUCUCAACAG44 212 UUCUCAGGAAUUUGUGUCUUU 44 213 GACAACUCUUU 44 214UCAGCUUCUGUUAGCCACUG 44 215 UGUUCAGCUUCUGUUAGCCACUGA 44 216CUGUUCAGCUUCUGUUAGCCACUGAUU 44 217 UUCUCAACAGAUCUGUCAAAUCGCCUGCAG 44 218GCCACUGAUUAAAUAUCUUUAUAUC 44 219 UCUGUUAGCCACUGAUUAAAUAUCUUUAUA 44 220GAGAAACUGUUCAGCUUCUGUUAGCCACUGA 44 221 UCUUUCUGAGAAACUGUUCAGCUUCUGUUAG44 222 CAGAUCUGUCAAAUCGCCUGCAGGUA 44 223 CAACAGAUCUGUCAAAUCGCCUGCAG 44224 AAACUGUUCAGCUUCUGUUAGCCACUGAUUAAA 44 225GAAACUGUUCAGCUUCUGUUAGCCACUGAUU 44 226 AAACUGUUCAGCUUCUGUUAGCCACUGA 44227 UGAGAAACUGUUCAGCUUCUGUUAGCCA 44 228 UUCUGAGAAACUGUUCAGCUUCUGUUAGCCAC44 229 UUCUGAGAAACUGUUCAGCUUCUGUU 44 230 GAUCUGUCAAAUCGCCUGCAGGUAA 44231 AUAAUGAAAACGCCGCCAUUUCUCA 44 232 AAACUGUUCAGCUUCUGUUAGCCAC 44 233UUGUGUCUUUCUGAGAAACUGUUCA 44 234 CCAAUUCUCAGGAAUUUGUGUCUUU 44 235AUCGCCUGCAGGUAAAAGCAUAUGG 44 236 UGAAAACGCCGCCAUUUCUCAACAGAUCUG 44 237CAUAAUGAAAACGCCGCCAUUUCUCAACAG 44 238 UGUUCAGCUUCUGUUAGCCACUGAUUAAAU 44239 CAGAUCUGUCAAAUCGCCUGCAGG 44 240 CAACAGAUCUGUCAAAUCGCCUGCAGG 44 241CUCAACAGAUCUGUCAAAUCGCCUGCAGG 44 242 GAUCUGUCAAAUCGCCUGCAGGU 44 243GAUCUGUCAAAUCGCCUGCAGG 44 244 GAUCUGUCAAAUCGCCUGCAG 44 245CAGAUCUGUCAAAUCGCCUGCAGGU 44 246 CAGAUCUGUCAAAUCGCCUGCAG 44 247GUGUCUUUCUGAGAAACUGUUCAGC 44 248 GAGAAACUGUUCAGCUUCUGUUAGCCAC 44 249GAAACUGUUCAGCUUCUGUUAGCCACUG 44 250 CUGUUCAGCUUCUGUUAGCCACUG 44 251AUCUGUCAAAUCGCCUGCAGGUAAAAG 44 252 GAUCUGUCAAAUCGCCUGCAGGUAAAAGC 44 253CACCGAUUGUCUUCGA 44 254 CCCUUGUACGAUUUAUG 44 255 UCUGUGUUUAAGGACUCU 45256 GCUGAAUUAUUUCUUCCCC 45 257 UUUUUCUGUCUGACAGCUG 45 258UCUGUUUUUGAGGAUUGC 45 259 CCACCGCAGAUUCAGGC 45 260 GCCCAAUGCCAUCCUGG 45261 UUUGCAGACCUCCUGCC 45 262 CAGUUUGCCGCUGCCCA 45 263GUUGCAUUCAAUGUUCUGAC 45 264 AUUUUUCCUGUAGAAUACUGG 45 265GCUGCCCAAUGCGAUCCUGGAGUUCCUGUAAGAU 45 266 GCUGCCCAAUGCCAUCCUGGAGUUCCUG45 267 GCUGCCCAAUGCCAUCCUGGAGUUCCUGUAA 45 268CAAUGCCAUCCUGGAGUUCCUGUAAGAUACC 45 269 GCUGCCCAAUGCCAUCCUGGAGUUCCUGUAAG45 270 CCAAUGCCAUCCUGGAGUUCCUGUAAGAUA 45 271UUGCCGCUGCCCAAUGCCAUCCUGGAGUUCCUGUA AGAU 45 272GCUGCCCAAUGCCAUCCUGGAGUUCCUGUAAGAU 45 273 CAAUGCCAUCCUGGAGUUCCUGUAAGA 45274 CAGUUUGCCGCUGCCCAAUGCCAUCC 45 275 CUUCCCCAGUUGCAUUCAAUGUUC 45 276CUGGCAUCUGUUUUUGAGGAUUG 45 277 UUAGAUCUGUCGCCCUACCU 45 278GCUGCCCAAUGCCAUCCUGGAGUUCCUGUAAGAUA CCAA 45 279GCCCAAUGCCAUCCUGGAGUUCCUGUAAGAUACC 45 280 CAUCCUGGAGUUCCUGUAAGAUACC 45281 UGCCAUCCUGGAGUUCCUGUAAGAUACC 45 282 UGCCAUCCUGGAGUUCCUGUAAGAU 45 283CAAUGCCAUCCUGGAGUUCCUGUAAGAU 45 284 GCCCAAUGCCAUCCUGGAGUUCCUGUAAGAU 45285 GCCCAAUGCCAUCCUGGAGUUCCUGUAA 45 286GCCGCUGCCCAAUGACAUCCUGGAGUUCCUGUAA 45 287 GCCAUCCUGGAGUUCCUGUAAGAUA 45288 CCAAUGCCAUCCUGGAGUUCCUGUA 45 289 CUGACAACAGUUUGCCGCUGCCCAA 45 290UUUGAGGAUUGCUGAAUUAUUUCUU 45 291 CAGUUUGCCGCUGCCCAAUGCCAUCCUGGA 45 292UUGCCGCUGCCCAAUGCCAUCCUGGAGUUC 45 293 UUUGCCGCUGCCCAAUGCCAUCCUG 45 294CCAAUGCCAUCCUGGAGUUCCU 45 295 CCCAAUGCCAUCCUGGAGUUCCUGUAAGA 45 296CCGCUGCCCAAUGCCAUCCUGGAGUUCC 45 297 CCCAAUGCCAUCCUGGAGUUCCUGUAAGAU 45298 CCGCUGCCCAAUGCCAUCCUGGAGUUCCUG 45 299 UGCCCAAUGCCAUCCUGGAGUUCCUGUAAG45 300 CCCAAUGCCAUCCUGGAGUUCCUGUAAG 45 301 UGCCCAAUGCCAUCCUGGAGUUCCUGUA45 302 CAAUGCCAUCCUGGAGUUCCUG 45 303 GCCGCUGCCCAAUGCCAUCCUGGAGUUCCUG 45304 AUUAGAUCUGUCGCCCUACCUCUUUUUUC 45 305 UGUCGCCCUACCUCUUUUUUCUGUCUG 45306 GCCCAAUGCCAUCCUGGAGUUCCUG 55 307 AGCCUCUCGCUCACUCACCCUGCAAAGGA 50308 CCACUCAGAGCUCAGAUCUUCUAACUUCC 50 309 CUUCCACUCAGAGCUCAGAUCUUCUAA 50310 GGGAUCCAGUAUACUUACAGGCUCC 50 311 CUCAGAGCUCAGAUCUU 50 312GGCUGCUUUGCCCUC 50 313 CUCAGAUCUUCUAACUUCCUCUUUAAC 50 314CUCAGAGCUCAGAUCUUCUAACUUCCUCU 50 315 CGCCUUCCACUCAGAGCUCAGAUCUUC 50 316UCAGCUCUUGAAGUAAACGGUUUACCG 50 317 UUUGCCCUCAGCUCUUGAAGUAAACGG 50 318GGCUGCUUUGCCCUCAGCUCUUGAAGU 50 319 CAGGAGCUAGGUCAGGCUGCUUUGCC 50 320UCCAAUAGUGGUCAGUCCAGGAGCU 50 321 AAAGAGAAUGGGAUCCAGUAUACUUAC 50 322AAAUAGCUAGAGCCAAAGAGAAUGGGA 50 323 GGCUGCUUUGCCCUCAGCUCUUGAAGUAAACGG 50324 AGGCUGCUUUGCCCUCAGCUCUUGAAGUAA 50 325 GUCAGGCUGCUUUGCCCUCAGCUCUUGAAG50 326 AGGUCAGGCUGCUUUGCCCUCAGCUCUUGA 50 327 CAGAGCUCAGAUCUUCUAACUUCCU50 328 CUUACAGGCUCCAAUAGUGGUCAGU 50 329 AUGGGAUCCAGUAUACUUACAGGCU 50 330AGAGAAUGGGAUCCAGUAUACUUAC 50 331 AACUUCCUCUUUAACAGAAAAGCAUAC 50 332GAGCCUCUCGCUCACUCACCCUGCAAAGGA 51 333 CUCAUACCUUCUGCUUGAUGAUC 51 334UCAAGGAAGAUGGCAUUUCU 51 335 GAAAGCCAGUCGGUAAGUUC 51 336 CACCCACCAUCACCC51 337 CCUCUGUGAUUUUAUAACUUGAU 51 338 UGAUAUCCUCAAGGUCACCC 51 339GGUACCUCCAACAUCAAGGAAGAUGGCAUU 51 340 AUUUCUAGUUUGGAGAUGGCAGUUUC 51 341CAUCAAGGAAGAUGGCAUUUCUAGUU 51 342 GAGCAGGUACCUCCAACAUCAAGGAA 51 343CUCCAACAUCAAGGAAGAUGGCAUUUCUAG 51 344 ACCAGAGUAACAGUCUGAGUAGGAG 51 345CACCAGAGUAACAGUCUGAGUAGGA 51 346 UCACCAGAGUAACAGUCUGAGUAGG 51 347GUCACCAGAGUAACAGUCUGAGUAG 51 348 ACCAGAGUAACAGUCUGAGUAGGAGC 51 349UUCUGUCCAAGCCCGGUUGAAAUC 51 350 ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG 51 351ACAUCAAGGAAGAUGGCAUUUCUAG 51 352 AUCAUUUUUUCUCAUACCUUCUGCU 51 353CACCCACCAUCACCCUCUGUG 51 354 AUCAUCUCGUUGAUAUCCUCAA 51 355CUCCAACAUCAAGGAAGAUGGCAUUUCU 51 356 CAUCAAGGAAGAUGGCAUUUCUAGU 51 357AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAA AA 52 358 UUGCUGGUCUUGUUUUUC 52 359CCGUAAUGAUUGUUCU 52 360 GCUGGUCUUGUUUUUCAA 52 361 UGGUCUUGUUUUUCAAAUUU52 362 GUCUUGUUUUUCAAAUUUUG 52 363 CUUGUUUUUCAAAUUUUGGG 52 364UGUUUUUCAAAUUUUGGGC 52 365 UCCAACUGGGGACGCCUCUGUUCCAAAUCCUGC 52 366UCCUGCAUUGUUGCCUGUAAG 52 367 UCCAACUGGGGACGCCUCUGUUCCAAAUCC 52 368ACUGGGGACGCCUCUGUUCCA 52 369 CCGUAAUGAUUGUUCUAGCC 52 370UGUUAAAAAACUUACUUCGA 53 371 CUGUUGCCUCCGGUUCUG 53 372 UUGGCUCUGGCCUGUCCU53 373 UUCAACUGUUGCCUCCGGUUCUGAAGGUGUUCU 53 374UACUUCAUCCCACUGAUUCUGAAUU 53 375 CUGAAGGUGUUCUUGUACUUCAUCC 53 376CUGUUGCCUCCGGUUCUGAAGGUGU 53 377 CUGUUGCCUCCGGUUCUGAAGGUGUUCUUG 53 378CAACUGUUGCCUCCGGUUCUGAAGGUGUUC 53 379 UUGCCUCCGGUUCUGAAGGUGUUCUUGUAC 53380 GUUGCCUCCGGUUCUGAAGGUGUUC 53 381 CUCCGGUUCUGAAGGUGUUCUUG 53 382CUCCGGUUCUGAAGGUGUUCUU 53 383 CUCCGGUUCUGAAGGUGUUCU 53 384CUCCGGUUCUGAAGGUGUUC 53 385 CUCCGGUUCUGAAGGUGUU 53 386CAUUCAACUGUUGCCUCCGGUUCUG 53 387 CUGUUGCCUCCGGUUCUGAAGGUG 53 388CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG 53 389 UACUAACCUUGGUUUCUGUGA 53 390UGUAUAGGGACCCUCCUUCCAUGACUC 53 391 CUAACCUUGGUUUCUGUGAUUUUCU 53 392GGUAUCUUUGAUACUAACCUUGGUUUC 53 393 AUUCUUUCAACUAGAAUAAAAG 53 394GAUUCUGAAUUCUUUCAACUAGAAU 53 395 AUCCCACUGAUUCUGAAUUC 53 396AACCGAGACCGGACAGGAUUCU 53 397 GGAAGCUAAGGAAGAAGCUGAGCAGG 55 398CUGUUGCAGUAAUCUAUGAG 55 399 UGCCAUUGUUUCAUCAGCUCUUU 55 400UGCAGUAAUCUAUGAGUUUC 55 401 UCCUGUAGGACAUUGGCAGU 55 131GAGUCUUCUAGGAGCCUU

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) targets a region of a DMD RNA (e.g., the Dp427mtranscript of SEQ ID NO: 2239). In some embodiments, an oligonucleotideuseful for targeting DMD (e.g., for exon skipping) comprises a region ofcomplementarity to a DMD RNA (e.g., the Dp427m transcript of SEQ ID NO:2239). In some embodiments, an oligonucleotide useful for targeting DMD(e.g., for exon skipping) comprises a region of complementarity to anexon of a DMD RNA (e.g., any one of SEQ ID NOs: 2240-2250). Examples ofDMD RNA sequences and exon sequences are provided below.

Homo sapiens dystrophin (DMD), transcript variant Dp427m, mRNA (NCBIReference Sequence: NM_004006.2)TCCTGGCATCAGTTACTGTGTTGACTCACTCAGTGTTGGGATCACTCACTTTCCCCCTACAGGACTCAGATCTGGGAGGCAATTACCTTCGGAGAAAAACGAATAGGAAAAACTGAAGTGTTACTTTTTTTAAAGCTGCTGAAGTTTGTTGGTTTCTCATTGTTTTTAAGCCTACTGGAGCAATAAAGTTTGAAGAACTTTTACCAGGTTTTTTTTATCGCTGCCTTGATATACACTTTTCAAAATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAATGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAGACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTCAACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAATCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGGCTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTTAATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCTATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATCAATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTACATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAGCACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACCACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATTGATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGGACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTACATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAAATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAGCTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGACTGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACGCCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGGTGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGATGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACATCCTTCTCAAATGGCAACGTCTTACTGAAGAACAGTGCCTTTTTAGTGCATGGCTTTCAGAAAAAGAAGATGCAGTGAACAAGATTCACACAACTGGCTTTAAAGATCAAAATGAAATGTTATCAAGTCTTCAAAAACTGGCCGTTTTAAAAGCGGATCTAGAAAAGAAAAAGCAATCCATGGGCAAACTGTATTCACTCAAACAAGATCTTCTTTCAACACTGAAGAATAAGTCAGTGACCCAGAAGACGGAAGCATGGCTGGATAACTTTGCCCGGTGTTGGGATAATTTAGTCCAAAAACTTGAAAAGAGTACAGCACAGATTTCACAGGCTGTCACCACCACTCAGCCATCACTAACACAGACAACTGTAATGGAAACAGTAACTACGGTGACCACAAGGGAACAGATCCTGGTAAAGCATGCTCAAGAGGAACTTCCACCACCACCTCCCCAAAAGAAGAGGCAGATTACTGTGGATTCTGAAATTAGGAAAAGGTTGGATGTTGATATAACTGAACTTCACAGCTGGATTACTCGCTCAGAAGCTGTGTTGCAGAGTCCTGAATTTGCAATCTTTCGGAAGGAAGGCAACTTCTCAGACTTAAAAGAAAAAGTCAATGCCATAGAGCGAGAAAAAGCTGAGAAGTTCAGAAAACTGCAAGATGCCAGCAGATCAGCTCAGGCCCTGGTGGAACAGATGGTGAATGAGGGTGTTAATGCAGATAGCATCAAACAAGCCTCAGAACAACTGAACAGCCGGTGGATCGAATTCTGCCAGTTGCTAAGTGAGAGACTTAACTGGCTGGAGTATCAGAACAACATCATCGCTTTCTATAATCAGCTACAACAATTGGAGCAGATGACAACTACTGCTGAAAACTGGTTGAAAATCCAACCCACCACCCCATCAGAGCCAACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGGATGAAGTCAACCGGCTATCAGGTCTTCAACCTCAAATTGAACGATTAAAAATTCAAAGCATAGCCCTGAAAGAGAAAGGACAAGGACCCATGTTCCTGGATGCAGACTTTGTGGCCTTTACAAATCATTTTAAGCAAGTCTTTTCTGATGTGCAGGCCAGAGAGAAAGAGCTACAGACAATTTTTGACACTTTGCCACCAATGCGCTATCAGGAGACCATGAGTGCCATCAGGACATGGGTCCAGCAGTCAGAAACCAAACTCTCCATACCTCAACTTAGTGTCACCGACTATGAAATCATGGAGCAGAGACTCGGGGAATTGCAGGCTTTACAAAGTTCTCTGCAAGAGCAACAAAGTGGCCTATACTATCTCAGCACCACTGTGAAAGAGATGTCGAAGAAAGCGCCCTCTGAAATTAGCCGGAAATATCAATCAGAATTTGAAGAAATTGAGGGACGCTGGAAGAAGCTCTCCTCCCAGCTGGTTGAGCATTGTCAAAAGCTAGAGGAGCAAATGAATAAACTCCGAAAAATTCAGAATCACATACAAACCCTGAAGAAATGGATGGCTGAAGTTGATGTTTTTCTGAAGGAGGAATGGCCTGCCCTTGGGGATTCAGAAATTCTAAAAAAGCAGCTGAAACAGTGCAGACTTTTAGTCAGTGATATTCAGACAATTCAGCCCAGTCTAAACAGTGTCAATGAAGGTGGGCAGAAGATAAAGAATGAAGCAGAGCCAGAGTTTGCTTCGAGACTTGAGACAGAACTCAAAGAACTTAACACTCAGTGGGATCACATGTGCCAACAGGTCTATGCCAGAAAGGAGGCCTTGAAGGGAGGTTTGGAGAAAACTGTAAGCCTCCAGAAAGATCTATCAGAGATGCACGAATGGATGACACAAGCTGAAGAAGAGTATCTTGAGAGAGATTTTGAATATAAAACTCCAGATGAATTACAGAAAGCAGTTGAAGAGATGAAGAGAGCTAAAGAAGAGGCCCAACAAAAAGAAGCGAAAGTGAAACTCCTTACTGAGTCTGTAAATAGTGTCATAGCTCAAGCTCCACCTGTAGCACAAGAGGCCTTAAAAAAGGAACTTGAAACTCTAACCACCAACTACCAGTGGCTCTGCACTAGGCTGAATGGGAAATGCAAGACTTTGGAAGAAGTTTGGGCATGTTGGCATGAGTTATTGTCATACTTGGAGAAAGCAAACAAGTGGCTAAATGAAGTAGAATTTAAACTTAAAACCACTGAAAACATTCCTGGCGGAGCTGAGGAAATCTCTGAGGTGCTAGATTCACTTGAAAATTTGATGCGACATTCAGAGGATAACCCAAATCAGATTCGCATATTGGCACAGACCCTAACAGATGGCGGAGTCATGGATGAGCTAATCAATGAGGAACTTGAGACATTTAATTCTCGTTGGAGGGAACTACATGAAGAGGCTGTAAGGAGGCAAAAGTTGCTTGAACAGAGCATCCAGTCTGCCCAGGAGACTGAAAAATCCTTACACTTAATCCAGGAGTCCCTCACATTCATTGACAAGCAGTTGGCAGCTTATATTGCAGACAAGGTGGACGCAGCTCAAATGCCTCAGGAAGCCCAGAAAATCCAATCTGATTTGACAAGTCATGAGATCAGTTTAGAAGAAATGAAGAAACATAATCAGGGGAAGGAGGCTGCCCAAAGAGTCCTGTCTCAGATTGATGTTGCACAGAAAAAATTACAAGATGTCTCCATGAAGTTTCGATTATTCCAGAAACCAGCCAATTTTGAGCAGCGTCTACAAGAAAGTAAGATGATTTTAGATGAAGTGAAGATGCACTTGCCTGCATTGGAAACAAAGAGTGTGGAACAGGAAGTAGTACAGTCACAGCTAAATCATTGTGTGAACTTGTATAAAAGTCTGAGTGAAGTGAAGTCTGAAGTGGAAATGGTGATAAAGACTGGACGTCAGATTGTACAGAAAAAGCAGACGGAAAATCCCAAAGAACTTGATGAAAGAGTAACAGCTTTGAAATTGCATTATAATGAGCTGGGAGCAAAGGTAACAGAAAGAAAGCAACAGTTGGAGAAATGCTTGAAATTGTCCCGTAAGATGCGAAAGGAAATGAATGTCTTGACAGAATGGCTGGCAGCTACAGATATGGAATTGACAAAGAGATCAGCAGTTGAAGGAATGCCTAGTAATTTGGATTCTGAAGTTGCCTGGGGAAAGGCTACTCAAAAAGAGATTGAGAAACAGAAGGTGCACCTGAAGAGTATCACAGAGGTAGGAGAGGCCTTGAAAACAGTTTTGGGCAAGAAGGAGACGTTGGTGGAAGATAAACTCAGTCTTCTGAATAGTAACTGGATAGCTGTCACCTCCCGAGCAGAAGAGTGGTTAAATCTTTTGTTGGAATACCAGAAACACATGGAAACTTTTGACCAGAATGTGGACCACATCACAAAGTGGATCATTCAGGCTGACACACTTTTGGATGAATCAGAGAAAAAGAAACCCCAGCAAAAAGAAGACGTGCTTAAGCGTTTAAAGGCAGAACTGAATGACATACGCCCAAAGGTGGACTCTACACGTGACCAAGCAGCAAACTTGATGGCAAACCGCGGTGACCACTGCAGGAAATTAGTAGAGCCCCAAATCTCAGAGCTCAACCATCGATTTGCAGCCATTTCACACAGAATTAAGACTGGAAAGGCCTCCATTCCTTTGAAGGAATTGGAGCAGTTTAACTCAGATATACAAAAATTGCTTGAACCACTGGAGGCTGAAATTCAGCAGGGGGTGAATCTGAAAGAGGAAGACTTCAATAAAGATATGAATGAAGACAATGAGGGTACTGTAAAAGAATTGTTGCAAAGAGGAGACAACTTACAACAAAGAATCACAGATGAGAGAAAGCGAGAGGAAATAAAGATAAAACAGCAGCTGTTACAGACAAAACATAATGCTCTCAAGGATTTGAGGTCTCAAAGAAGAAAAAAGGCTCTAGAAATTTCTCATCAGTGGTATCAGTACAAGAGGCAGGCTGATGATCTCCTGAAATGCTTGGATGACATTGAAAAAAAATTAGCCAGCCTACCTGAGCCCAGAGATGAAAGGAAAATAAAGGAAATTGATCGGGAATTGCAGAAGAAGAAAGAGGAGCTGAATGCAGTGCGTAGGCAAGCTGAGGGCTTGTCTGAGGATGGGGCCGCAATGGCAGTGGAGCCAACTCAGATCCAGCTCAGCAAGCGCTGGCGGGAAATTGAGAGCAAATTTGCTCAGTTTCGAAGACTCAACTTTGCACAAATTCACACTGTCCGTGAAGAAACGATGATGGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCACAAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAAGAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACAGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGGAAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGTTCAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCTCTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAGGCAACAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAAGAACTCATTACCGCTGCCCAAAATTTGAAAAACAAGACCAGCAATCAAGAGGCTAGAACAATCATTACGGATCGAATTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTTGAATGAAATGTTAAAGGATTCAACACAATGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCCCTATACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAGCAGTTGGCCAAAGACCTCCGCCAGTGGCAGACAAATGTAGATGTGGCAAATGACTTGGCCCTGAAACTTCTCCGGGATTATTCTGCAGATGATACCAGAAAAGTCCACATGATAACAGAGAATATCAATGCCTCTTGGAGAAGCATTCATAAAAGGGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAACTCATAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGGCAAGACCTCCAAGGTGAAATTGAAGCTCACACAGATGTTTATCACAACCTGGATGAAAACAGCCAAAAAATCCTGAGATCCCTGGAAGGTTCCGATGATGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAGTGAACTTCGGAAAAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGCGTCTGCACCTTTCTCTGCAGGAACTTCTGGTGTGGCTACAGCTGAAAGATGATGAATTAAGCCGGCAGGCACCTATTGGAGGCGACTTTCCAGCAGTTCAGAAGCAGAACGATGTACATAGGGCCTTCAAGAGGGAATTGAAAACTAAAGAACCTGTAATCATGAGTACTCTTGAGACTGTACGAATATTTCTGACAGAGCAGCCTTTGGAAGGACTAGAGAAACTCTACCAGGAGCCCAGAGAGCTGCCTCCTGAGGAGAGAGCCCAGAATGTCACTCGGCTTCTACGAAAGCAGGCTGAGGAGGTCAATACTGAGTGGGAAAAATTGAACCTGCACTCCGCTGACTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCAGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATGTAGGAAGTCTTTTCCACATGGCAGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCATGGTTTTTATAATATTCATACAACAAAGAGGATTAGACAGTAAGAGTTTACAAGAAATAAATCTATATTTTTGTGAAGGGTAGTGGTATTATACTGTAGATTTCAGTAGTTTCTAAGTCTGTTATTGTTTTGTTAACAATGGCAGGTTTTACACGTCTATGCAATTGTACAAAAAAGTTATAAGAAAACTACATGTAAAATCTTGATAGCTAAATAACTTGCCATTTCTTTATATGGAACGCATTTTGGGTTGTTTAAAAATTTATAACAGTTATAAAGAAAGATTGTAAACTAAAGTGTGCTTTATAAAAAAAAGTTGTTTATAAAAACCCCTAAAAACAAAACAAACACACACACACACACATACACACACACACACAAAACTTTGAGGCAGCGCATTGTTTTGCATCCTTTTGGCGTGATATCCATATGAAATTCATGGCTTTTTCTTTTTTTGCATATTAAAGATAAGACTTCCTCTACCACCACACCAAATGACTACTACACACTGCTCATTTGAGAACTGTCAGCTGAGTGGGGCAGGCTTGAGTTTTCATTTCATATATCTATATGTCTATAAGTATATAAATACTATAGTTATATAGATAAAGAGATACGAATTTCTATAGACTGACTTTTTCCATTTTTTAAATGTTCATGTCACATCCTAATAGAAAGAAATTACTTCTAGTCAGTCATCCAGGCTTACCTGCTTGGTCTAGAATGGATTTTTCCCGGAGCCGGAAGCCAGGAGGAAACTACACCACACTAAAACATTGTCTACAGCTCCAGATGTTTCTCATTTTAAACAACTTTCCACTGACAACGAAAGTAAAGTAAAGTATTGGATTTTTTTAAAGGGAACATGTGAATGAATACACAGGACTTATTATATCAGAGTGAGTAATCGGTTGGTTGGTTGATTGATTGATTGATTGATACATTCAGCTTCCTGCTGCTAGCAATGCCACGATTTAGATTTAATGATGCTTCAGTGGAAATCAATCAGAAGGTATTCTGACCTTGTGAACATCAGAAGGTATTTTTTAACTCCCAAGCAGTAGCAGGACGATGATAGGGCTGGAGGGCTATGGATTCCCAGCCCATCCCTGTGAAGGAGTAGGCCACTCTTTAAGTGAAGGATTGGATGATTGTTCATAATACATAAAGTTCTCTGTAATTACAACTAAATTATTATGCCCTCTTCTCACAGTCAAAAGGAACTGGGTGGTTTGGTTTTTGTTGCTTTTTTAGATTTATTGTCCCATGTGGGATGAGTTTTTAAATGCCACAAGACATAATTTAAAATAAATAAACTTTGGGAAAAGGTGTAAAACAGTAGCCCCATCACATTTGTGATACTGACAGGTATCAACCCAGAAGCCCATGAACTGTGTTTCCATCCTTTGCATTTCTCTGCGAGTAGTTCCACACAGGTTTGTAAGTAAGTAAGAAAGAAGGCAAATTGATTCAAATGTTACAAAAAAACCCTTCTTGGTGGATTAGACAGGTTAAATATATAAACAAACAAACAAAAATTGCTCAAAAAAGAGGAGAAAAGCTCAAGAGGAAAAGCTAAGGACTGGTAGGAAAAAGCTTTACTCTTTCATGCCATTTTATTTCTTTTTGATTTTTAAATCATTCATTCAATAGATACCACCGTGTGACCTATAATTTTGCAAATCTGTTACCTCTGACATCAAGTGTAATTAGCTTTTGGAGAGTGGGCTGACATCAAGTGTAATTAGCTTTTGGAGAGTGGGTTTTGTCCATTATTAATAATTAATTAATTAACATCAAACACGGCTTCTCATGCTATTTCTACCTCACTTTGGTTTTGGGGTGTTCCTGATAATTGTGCACACCTGAGTTCACAGCTTCACCACTTGTCCATTGCGTTATTTTCTTTTTCCTTTATAATTCTTTCTTTTTCCTTCATAATTTTCAAAAGAAAACCCAAAGCTCTAAGGTAACAAATTACCAAATTACATGAAGATTTGGTTTTTGTCTTGCATTTTTTTCCTTTATGTGACGCTGGACCTTTTCTTTACCCAAGGATTTTTAAAACTCAGATTTAAAACAAGGGGTTACTTTACATCCTACTAAGAAGTTTAAGTAAGTAAGTTTCATTCTAAAATCAGAGGTAAATAGAGTGCATAAATAATTTTGTTTTAATCTTTTTGTTTTTCTTTTAGACACATTAGCTCTGGAGTGAGTCTGTCATAATATTTGAACAAAAATTGAGAGCTTTATTGCTGCATTTTAAGCATAATTAATTTGGACATTATTTCGTGTTGTGTTCTTTATAACCACCAAGTATTAAACTGTAAATCATAATGTAACTGAAGCATAAACATCACATGGCATGTTTTGTCATTGTTTTCAGGTACTGAGTTCTTACTTGAGTATCATAATATATTGTGTTTTAACACCAACACTGTAACATTTACGAATTATTTTTTTAAACTTCAGTTTTACTGCATTTTCACAACATATCAGACTTCACCAAATATATGCCTTACTATTGTATTATAGTACTGCTTTACTGTGTATCTCAATAAAGCACGCAGTTATGTTAC (SEQ ID NO: 2239)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 8(nucleotide positions 894-1075 of NCBI Reference Sequence: NM_004006.2)ATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTACATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAG (SEQ ID NO: 2240)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 23(nucleotide positions 3194-3406 of NCBI Reference Sequence: NM_004006.2)GCTTTACAAAGTTCTCTGCAAGAGCAACAAAGTGGCCTATACTATCTCAGCACCACTGTGAAAGAGATGTCGAAGAAAGCGCCCTCTGAAATTAGCCGGAAATATCAATCAGAATTTGAAGAAATTGAGGGACGCTGGAAGAAGCTCTCCTCCCAGCTGGTTGAGCATTGTCAAAAGCTAGAGGAGCAAATGAATAAACTCCGAAAAATTCAG (SEQ ID NO: 2241)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 43(nucleotide positions 6362-6534 of NCBI Reference Sequence: NM_004006.2)AATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACAGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGGAAAAAGTTAACAAAATGTACAAGGACCGACAAGG (SEQ ID NO: 2242)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 44(nucleotide positions 6535-6682 of NCBI Reference Sequence: NM_004006.2)GCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAG (SEQ ID NO: 2243)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 45(nucleotide positions 6683-6858 of NCBI Reference Sequence: NM_004006.2)GAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAG (SEQ ID NO: 2244)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 46(nucleotide positions 6859-7006 of NCBI Reference Sequence: NM_004006.2)GCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAG (SEQ ID NO: 2245)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 50(nucleotide positions 7445-7553 of NCBI Reference Sequence: NM_004006.2)AGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCT (SEQ ID NO: 2246)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 51(nucleotide positions 7554-7786 of NCBI Reference Sequence: NM_004006.2)CTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAG (SEQ ID NO: 2247)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 52(nucleotide positions 7787-7904 of NCBI Reference Sequence: NM_004006.2)GCAACAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAAGAACTCATTACCGCTGCCCAAAATTTGAAAAACAAGACCAGCAATCAAGAGGCTAGAACAATCATTACGGATCGAA (SEQ ID NO: 2248)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 53(nucleotide positions 7905-8116 of NCBI Reference Sequence: NM_004006.2)TTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTTGAATGAAATGTTAAAGGATTCAACACAATGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCCCTATACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAG (SEQ ID NO: 2249)Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 55(nucleotide positions 8272-8461 of NCBI Reference Sequence: NM_004006.2)GGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAACTCATAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGGCAA (SEQ IDNO: 2250)

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) targets an exonic splicing enhancer (ESE) sequence inDMD (e.g., an ESE sequence of exon 23, 44, 45, 46, 50, 51, 52, 53, or55). In some embodiments, an oligonucleotide useful for targeting DMD(e.g., for exon skipping) targets an exonic splicing enhancer (ESE)sequence in DMD (e.g., an ESE sequence of exon 8, 23, 43, 44, 45, 46,50, 51, 52, 53, or 55). In some embodiments, an oligonucleotide usefulfor targeting DMD (e.g., for exon skipping) targets an ESE sequence ofDMD exon 51 (e.g., the ESEs listed in Table 15). In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping)targets an ESE sequence of DMD exon 8, 23, 42, 44, 45, 46, 50, 52, 53,or 55 (e.g., an ESE listed in Table 11).

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping, such as for skipping one or more of exons 8, 23, 42,44, 45, 46, 50, 52, 53, and 55) comprises a region of complementarity toa target sequence comprising one or more full or partial ESEs of a DMDtranscript (e.g., one or more full or partial ESEs listed in Table 15 orTable 11). In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 402-436 and 2043-2238. In someembodiments, the oligonucleotide comprises a region of complementarityto a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:402-436 and 2043-2238.

TABLE 15 Exonic splicing enhancers within exon 51 of DMD Ref. start SEQESE Motif ESE # position* ID NO: Sequence Name 51-1 1565421 402 CTACTCADp427m_51:SRSF5 51-2 1565426 403 CAGACTG Dp427m_51:SRSF1 (IgM-BRCA1)51-3 1565428 404 GACTGTTA Dp427m_51:SRSF2 51-4 1565433 405 TTACTCTDp427m_51:SRSF5 51-5 1565435 406 ACTCTGG Dp427m_51:SRSF5 51-6 1565436407 CTCTGGT Dp427m_51:SRSF1 (IgM-BRCA1) 51-7 1565442 408 TGACACADp427m_51:SRSF5 51-8 1565443 409 GACACAA Dp427m_51:SRSF1 51-9 1565444410 ACACAAC Dp427m_51:SRSF5 51-10 1565448 411 AACCTGTG Dp427m_51:SRSF251-11 1565450 412 CCTGTGG Dp427m_51:SRSF5 51-12 1565451 413 CTGTGGTDp427m_51:SRSF1 (IgM-BRCA1) 51-13 1565455 414 GGTTACTA Dp427m_51:SRSF251-14 1565457 415 TTACTAA Dp427m_51:SRSF5 51-15 1565460 416 CTAAGGADp427m_51:SRSF1 (IgM-BRCA1) 51-16 1565469 417 CTGCCATDp427m_51:SRSF1 (IgM-BRCA1) 51-17 1565479 418 CAAACTADp427m_51:SRSF1 (IgM-BRCA1) 51-18 1565508 419 TGGAGGT Dp427m_51:SRSF151-19 1565512 420 GGTACCTG Dp427m_51:SRSF2 51-20 1565528 421 GATTTCAADp427m_51:SRSF2 51-21 1565530 422 TTTCAAC Dp427m_51:SRSF5 51-22 1565532423 TCAACCG Dp427m_51:SRSF5 51-23 1565533 424 CAACCGGDp427m_51:SRSF1 (IgM-BRCA1) 51-24 1565544 425 GGACAGAA Dp427m_51:SRSF251-25 1565556 426 CCGACTG Dp427m_51:SRSF1 (IgM-BRCA1) 51-26 1565557 427CGACTGG Dp427m_51:SRSF5 51-27 1565565 428 TTTCTCTG Dp427m_51:SRSF2 51-281565567 429 TCTCTGC Dp427m_51:SRSF5 51-29 1565591 430 TCACAGADp427m_51:SRSF5 51-30 1565592 431 CACAGA Dp427m_51:SRSF6 51-31 1565593432 ACAGAGG Dp427m_51:SRSF5 51-32 1565594 433 CAGAGGGDp427m_51:SRSF1 (IgM-BRCA1) 51-33 1565615 434 CTTGAGG Dp427m_51:SRSF551-34 1565617 435 TGAGGA Dp427m_51:SRSF6 51-35 1565630 436 GAGATGADp427m_51:SRSFl *Ref. start position refers to the position of the firstnucleotide of the ESE motif in nucleotides 5,001-2,225,382 of NCBIReference Sequence NG_012232.1 (NG_012232, version 1). Nucleotides5,001-2,225,382 of NCBI Reference Sequence NG_012232.1 (NG_012232,version 1) correspond to Homo sapiens dystrophin (DMD) gene onchromosome X.

TABLE 11Exonic splicing enhancers within exons 8, 23, 43, 44, 45, 46, 50, 52, 53,and 55 of DMD Ref. start SEQ ID ESE Motif Exon position* NO: SequenceName  8  640346 2047 TCCATC Dp427m_8:SRSF6  8  640358 2048 TACATCDp427m_8:SRSF6  8  640362 2049 TCACATC Dp427m_8:SRSF5  8  640363 2050CACATC Dp427m_8:SRSF6  8  640367 2051 TCACTCT Dp427m_8:SRSF5  8  6403682052 CACTCTT Dp427m_8:SRSF1 (IgM-BRCA1)  8  640373 2053 TTCCAAGDp427m_8:SRSF5  8  640383 2054 TGCCTC Dp427m_8:SRSF6  8  640385 2055CCTCAAC Dp427m_8:SRSF5  8  640388 2056 CAACAAG Dp427m_8:SRSF5  8  6404342057 GGCCACCT Dp427m_8:SRSF2  8  640439 2058 CCTAAAG Dp427m_8:SRSF5  8 640447 2059 GACTAAAG Dp427m_8:SRSF2  8  640469 2060 TTACATCDp427m_8:SRSF5  8  640470 2048 TACATC Dp427m_8:SRSF6  8  640477 2061TCAAATG Dp427m_8:SRSF5  8  640490 2062 TCTCAAC Dp427m_8:SRSF5 23  8709032063 TTACAAA Dp427m_23:SRSF5 23  870910 2064 GTTCTCTG Dp427m_23:SRSF2 23 870912  429 TCTCTGC Dp427m_23:SRSF5 23  870933 2065 GGCCTATADp427m_23:SRSF2 23  870944 2066 TCTCAGC Dp427m_23:SRSF5 23  870945 2067CTCAGCA Dp427m_23:SRSF1 (IgM-BRCA1) 23  870950 2068 CACCACTGDp427m_23:SRSF2 23  870952 2069 CCACTGT Dp427m_23:SRSF5 23  870970 2070CGAAGAA Dp427m_23:SRSF1 (IgM-BRCA1) 23  870979 2071 CGCCCTCDp427m_23:SRSF1 (IgM-BRCA1) 23  870980 2072 GCCCTCTG Dp427m_23:SRSF2 23 870993 2073 AGCCGGA Dp427m_23:SRSF1 (IgM-BRCA1) 23  871014 2074 TTTGAAGDp427m_23:SRSF5 23  871028 2075 GGGACGC Dp427m_23:SRSF1 23  871029 2076GGACGCTG Dp427m_23:SRSF2 23  871032 2077 CGCTGGADp427m_23:SRSF1 (IgM-BRCA1) 23  871049 2078 CTCCCAGDp427m_23:SRSF1 (IgM-BRCA1) 23  871050 2079 TCCCAGC Dp427m_23:SRSF5 23 871051 2080 CCCAGCT Dp427m_23:SRSF1 (IgM-BRCA1) 23  871053 2081 CAGCTGGDp427m_23:SRSF1 (IgM-BRCA1) 23  871070 2082 TCAAAAG Dp427m_23:SRSF5 23 871077 2083 CTAGAGG Dp427m_23:SRSF5 23  871078 2084 TAGAGGADp427m_23:SRSF1 23  871090 2085 TGAATA Dp427m_23:SRSF6 23  871098 2086CTCCGAA Dp427m_23:SRSF1 (IgM-BRCA1) 43 1051912 2087 ATAAAAGDp427m_43:SRSF5 43 1051922 2088 GTCTACAA Dp427m_43:SRSF2 43 1051924 2089CTACAAC Dp427m_43:SRSF5 43 1051929 2090 ACAAAGC Dp427m_43:SRSF5 431051934 2091 GCTCAGG Dp427m_43:SRSF5 43 1051935 2092 CTCAGGTDp427m_43:SRSF1 (IgM-BRCA1) 43 1051955 2093 TTCATA Dp427m_43:SRSF6 431051956 2094 TCATAGC Dp427m_43:SRSF5 43 1051967 2095 AGACAGCDp427m_43:SRSF5 43 1051971 2096 AGCAGC Dp427m_43:SRSF6 43 1051986 2097TGCAAC Dp427m_43:SRSF6 43 1051991 2098 CGCCTGTG Dp427m_43:SRSF2 431051993  412 CCTGTGG Dp427m_43:SRSF5 43 1051994 2099 CTGTGGADp427m_43:SRSF1 (IgM-BRCA1) 43 1051995 2100 TGTGGA Dp427m_43:SRSF6 431052009 2101 AGCTACAG Dp427m_43:SRSF2 43 1052011 2102 CTACAGGDp427m_43:SRSF5 43 1052012 2103 TACAGGA Dp427m_43:SRSF1 (IgM-BRCA1) 431052022 2104 TCTCTCC Dp427m_43:SRSF5 43 1052023 2105 CTCTCCCADp427m_43:SRSF2 43 1052025 2078 CTCCCAG Dp427m_43:SRSF1 (IgM-BRCA1) 431052026 2079 TCCCAGC Dp427m_43:SRSF5 43 1052027 2080 CCCAGCTDp427m_43:SRSF1 (IgM-BRCA1) 43 1052035 2106 GATTTCCA Dp427m_43:SRSF2 431052040 2107 CCAATGG Dp427m_43:SRSF5 43 1052064 2108 GTACAAGDp427m_43:SRSF5 43 1052071 2109 GACCGACA Dp427m_43:SRSF2 43 1052073 2110CCGACAA Dp427m_43:SRSF1 (IgM-BRCA1) 43 1052074 2111 CGACAAGDp427m_43:SRSF5 44 1122553 2112 TGACAGA Dp427m_44:SRSF5 44 1122575 2113CGGCGTT Dp427m_44:SRSF1 (IgM-BRCA1) 44 1122607 2114 TCAGTGGDp427m_44:SRSF5 44 1122612 2115 GGCTAACA Dp427m_44:SRSF2 44 1122617 2116ACAGAAG Dp427m_44:SRSF5 44 1122634 2117 TCTCAGA Dp427m_44:SRSF5 441122635 2118 CTCAGAA Dp427m_44:SRSF1 (IgM-BRCA1) 44 1122643  409 GACACAADp427m_44:SRSF1 44 1122649 2119 AATTCCTG Dp427m_44:SRSF2 44 1122654 2120CTGAGAA Dp427m_44:SRSF1 (IgM-BRCA1) 44 1122685 2121 GTATCTTADp427m_44:SRSF2 45 1371096 2122 GAACTCCA Dp427m_45:SRSF2 45 1371097 2123AACTCCAG Dp427m_45:SRSF2 45 1371099 2124 CTCCAGG Dp427m_45:SRSF5 451371117 2125 CAGCGGC Dp427m_45:SRSF1 (IgM-BRCA1) 45 1371118 2126 AGCGGCDp427m_45:SRSF6 45 1371133 2127 TCAGAAC Dp427m_45:SRSF5 45 1371136 2128GAACATTG Dp427m_45:SRSF2 45 1371142 2129 TGAATGC Dp427m_45:SRSF5 451371143 2130 GAATGCAA Dp427m_45:SRSF2 45 1371146 2097 TGCAACDp427m_45:SRSF6 45 1371148 2131 CAACTGG Dp427m_45:SRSF5 45 1371151 2132CTGGGGA Dp427m_45:SRSF1 (IgM-BRCA1) 45 1371165 2133 ATTCAGCDp427m_45:SRSF5 45 1371188 2134 TGCCAGTA Dp427m_45:SRSF2 45 1371193 2135GTATTCTA Dp427m_45:SRSF2 45 1371198 2102 CTACAGG Dp427m_45:SRSF5 451371199 2103 TACAGGA Dp427m_45:SRSF1 (IgM-BRCA1) 45 1371220 2136 TGAATCDp427m_45:SRSF6 45 1371225 2137 CTGCGGT Dp427m_45:SRSF1 (IgM-BRCA1) 451371226 2138 TGCGGT Dp427m_45:SRSF6 45 1371228 2139 CGGTGGCDp427m_45:SRSF1 (IgM-BRCA1) 45 1371231 2140 TGGCAGG Dp427m_45:SRSF5 451371232 2141 GGCAGGA Dp427m_45:SRSF1 (IgM-BRCA1) 45 1371235 2142 AGGAGGTDp427m_45:SRSF1 45 1371239 2143 GGTCTGCA Dp427m_45:SRSF2 45 1371240 2144GTCTGCAA Dp427m_45:SRSF2 45 1371249 2145 CAGCTGTDp427m_45:SRSF1 (IgM-BRCA1) 45 1371256 2146 CAGACAGDp427m_45:SRSF1 (IgM-BRCA1) 46 1407384 2147 CTAGAAG Dp427m_46:SRSF5 461407392 2148 ACAAAAG Dp427m_46:SRSF5 46 1407438 2149 GTTTTATGDp427m_46:SRSF2 46 1407440 2150 TTTATGG Dp427m_46:SRSF5 46 1407445 2151GGTTGGAG Dp427m_46:SRSF2 46 1407448 2152 TGGAGGADp427m_46:SRSF1 (IgM-BRCA1) 46 1407472 2153 GTATCCCA Dp427m_46:SRSF2 461407476 2154 CCCACTT Dp427m_46:SRSF1 (IgM-BRCA1) 46 1407477 2155 CCACTTGDp427m_46:SRSF5 46 1407478 2156 CACTTGA Dp427m_46:SRSF1 (IgM-BRCA1) 461407496 2096 AGCAGC Dp427m_46:SRSF6 46 1407504 2157 CTAAAAGDp427m_46:SRSF5 46 1407524 2158 AGTCAAG Dp427m_46:SRSF5 50 1519533 2159TTAGAAG Dp427m_50:SRSF5 50 1519539 2160 GATCTGAG Dp427m_50:SRSF2 501519541 2161 TCTGAGC Dp427m_50:SRSF5 50 1519542 2162 CTGAGCTDp427m_50:SRSF1 (IgM-BRCA1) 50 1519544 2163 GAGCTCTG Dp427m_50:SRSF2 501519549 2164 CTGAGTG Dp427m_50:SRSF1 (IgM-BRCA1) 50 1519550 2165 TGAGTGGDp427m_50:SRSF5 50 1519572 2166 TTACTTC Dp427m_50:SRSF5 50 1519573 2167TACTTC Dp427m_50:SRSF6 50 1519575 2168 CTTCAAG Dp427m_50:SRSF5 501519584 2169 CTGAGGG Dp427m_50:SRSF1 (IgM-BRCA1) 50 1519594 2096 AGCAGCDp427m_50:SRSF6 50 1519596 2170 CAGCCTG Dp427m_50:SRSF1 (IgM-BRCA1) 501519600 2171 CTGACCT Dp427m_50:SRSF1 (IgM-BRCA1) 50 1519607 2172AGCTCCTG Dp427m_50:SRSF2 50 1519609 2173 CTCCTGG Dp427m_50:SRSF5 501519617 2174 CTGACCA Dp427m_50:SRSF1 (IgM-BRCA1) 50 1519619 2175GACCACTA Dp427m_50:SRSF2 50 1519621 2176 CCACTAT Dp427m_50:SRSF5 501519624 2177 CTATTGG Dp427m_50:SRSF5 52 1609869 2178 TGCAGGDp427m_52:SRSF6 52 1609880 2179 GAACAGAG Dp427m_52:SRSF2 52 1609882  432ACAGAGG Dp427m_52:SRSF5 52 1609883 2180 CAGAGGCDp427m_52:SRSF1 (IgM-BRCA1) 52 1609887 2181 GGCGTC Dp427m_52:SRSF6 521609889 2182 CGTCCCCA Dp427m_52:SRSF2 52 1609890 2183 GTCCCCAGDp427m_52:SRSF2 52 1609892 2184 CCCCAGT Dp427m_52:SRSF1 (IgM-BRCA1) 521609893 2185 CCCAGTT Dp427m_52:SRSF1 (IgM-BRCA1) 52 1609911 2186 TTACCGCDp427m_52:SRSF5 52 1609912 2187 TACCGCTG Dp427m_52:SRSF2 52 1609917 2188CTGCCCA Dp427m_52:SRSF1 (IgM-BRCA1) 52 1609939 2189 GACCAGCADp427m_52:SRSF2 52 1609954 2190 GGCTAGAA Dp427m_52:SRSF2 52 1609969 2191TACGGA Dp427m_52:SRSF6 52 1609972 2192 GGATCGAA Dp427m_52:SRSF2 531660030 2193 GAATTCAG Dp427m_53:SRSF2 53 1660040 2114 TCAGTGGDp427m_53:SRSF5 53 1660041 2194 CAGTGGG Dp427m_53:SRSF1 (IgM-BRCA1) 531660053 2108 GTACAAG Dp427m_53:SRSF5 53 1660067 2127 TCAGAACDp427m_53:SRSF5 53 1660071 2195 AACCGGA Dp427m_53:SRSF1 53 1660074 2196CGGAGGC Dp427m_53:SRSF1 (IgM-BRCA1) 53 1660098 2197 TTAAAGGDp427m_53:SRSF5 53 1660103 2198 GGATTCAA Dp427m_53:SRSF2 53 1660112 2199ACAATGG Dp427m_53:SRSF5 53 1660117 2200 GGCTGGAA Dp427m_53:SRSF2 531660126  416 CTAAGGA Dp427m_53:SRSF1 (IgM-BRCA1) 53 1660138 2201 CTGAGCADp427m_53:SRSF1 (IgM-BRCA1) 53 1660141 2202 AGCAGGTDp427m_53:SRSF1 (IgM-BRCA1) 53 1660147 2203 TCTTAGG Dp427m_53:SRSF5 531660148 2204 CTTAGGA Dp427m_53:SRSF1 (IgM-BRCA1) 53 1660152 2205 GGACAGGDp427m_53:SRSF5 53 1660153 2206 GACAGGC Dp427m_53:SRSF1 53 1660157 2207GGCCAGAG Dp427m_53:SRSF2 53 1660159 2208 CCAGAGC Dp427m_53:SRSF5 531660172 2209 TGAGTC Dp427m_53:SRSF6 53 1660183 2210 AGGAGGGDp427m_53:SRSF1 53 1660188 2211 GGTCCCTA Dp427m_53:SRSF2 53 1660197 2212ACAGTAG Dp427m_53:SRSF5 53 1660211 2213 CCAAAAG Dp427m_53:SRSF5 531660222  430 TCACAGA Dp427m_53:SRSF5 53 1660223  431 CACAGADp427m_53:SRSF6 55 1711758 2214 CGAGAGG Dp427m_55:SRSF5 55 1711763 2215GGCTGCTT Dp427m_55:SRSF2 55 1711786 2216 GATTACTG Dp427m_55:SRSF2 551711788 2217 TTACTGC Dp427m_55:SRSF5 55 1711792 2097 TGCAACDp427m_55:SRSF6 55 1711802 2218 CCCCCTG Dp427m_55:SRSF1 (IgM-BRCA1) 551711803 2219 CCCCTGG Dp427m_55:SRSF5 55 1711804 2220 CCCTGGADp427m_55:SRSF1 (IgM-BRCA1) 55 1711820 2221 GTTTCTTG Dp427m_55:SRSF2 551711821 2222 TTTCTTG Dp427m_55:SRSF5 55 1711826 2223 TGCCTGGDp427m_55:SRSF5 55 1711831 2224 GGCTTACA Dp427m_55:SRSF2 55 1711834 2225TTACAGA Dp427m_55:SRSF5 55 1711835 2226 TACAGA Dp427m_55:SRSF6 551711836 2116 ACAGAAG Dp427m_55:SRSF5 55 1711852 2227 CTGCCAADp427m_55:SRSF1 (IgM-BRCA1) 55 1711853 2228 TGCCAATG Dp427m_55:SRSF2 551711860 2229 GTCCTACA Dp427m_55:SRSF2 55 1711863 2102 CTACAGGDp427m_55:SRSF5 55 1711864 2103 TACAGGA Dp427m_55:SRSF1 (IgM-BRCA1) 551711868 2230 GGATGCTA Dp427m_55:SRSF2 55 1711873 2231 CTACCCGDp427m_55:SRSF5 55 1711874 2232 TACCCGTA Dp427m_55:SRSF2 55 1711888 2233GGCTCCTA Dp427m_55:SRSF2 55 1711893 2147 CTAGAAG Dp427m_55:SRSF5 551711898 2234 AGACTCC Dp427m_55:SRSF5 55 1711899 2235 GACTCCAADp427m_55:SRSF2 55 1711901 2236 CTCCAAG Dp427m_55:SRSF5 55 1711903 2237CCAAGGG Dp427m_55:SRSF1 (IgM-BRCA1) 55 1711920 2238 CTGATGADp427m_55:SRSF1 (IgM-BRCA1) 55 1711928 2199 ACAATGG Dp427m_55:SRSF5*Ref. start position refers to the position of the first nucleotide ofthe ESE motif in nucleotides 5,001-2,225,382 of NCBI Reference SequenceNG_012232.1 (NG_012232, version 1). Nucleotides 5,001-2,225,382 of NCBIReference Sequence NG_012232.1 (NG_012232, version 1) correspond to Homosapiens dystrophin (DMD) gene on chromosome X.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 8. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 8. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 2047-2062. In some embodiments,the oligonucleotide comprises a region of complementarity to a targetsequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutivenucleotides of an ESE as set forth in any one of SEQ ID NOs: 2047-2062.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon8. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 2047-2062.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 2047-2062. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 2047-2062. In some embodiments, an oligonucleotide usefulfor targeting DMD (e.g., for exon skipping) is 20 nucleotides in length,and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 2047-2062. In someembodiments, an oligonucleotide useful for targeting DMD (e.g., for exonskipping) is 30 nucleotides in length, and comprises a region ofcomplementarity to a target sequence comprising at least 4 (e.g., 4, 5,6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one ofSEQ ID NOs: 2047-2062.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 23. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 23. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 429 and 2063-2086. In someembodiments, the oligonucleotide comprises a region of complementarityto a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:429 and 2063-2086.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon23. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 429 and 2063-2086.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 429 and 2063-2086. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 429 and 2063-2086. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:429 and 2063-2086. In some embodiments, an oligonucleotide useful fortargeting DMD (e.g., for exon skipping) is 30 nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 429 and 2063-2086.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 43. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 43. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 412, 2078-2080, and 2087-2111.In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 4 (e.g., 4, 5,6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one ofSEQ ID NOs: 412, 2078-2080, and 2087-2111.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon43. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 412, 2078-2080, and 2087-2111.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111. In someembodiments, an oligonucleotide useful for targeting DMD (e.g., for exonskipping) is 20-30 (e.g., 20, 25, 30) nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111. Insome embodiments, an oligonucleotide useful for targeting DMD (e.g., forexon skipping) is 20 nucleotides in length, and comprises a region ofcomplementarity to a target sequence comprising at least 4 (e.g., 4, 5,6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one ofSEQ ID NOs: 412, 2078-2080, and 2087-2111. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:412, 2078-2080, and 2087-2111.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 44. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 44. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 409 and 2112-2121. In someembodiments, the oligonucleotide comprises a region of complementarityto a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:409 and 2112-2121.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon44. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 409 and 2112-2121.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 409 and 2112-2121. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 409 and 2112-2121. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:409 and 2112-2121. In some embodiments, an oligonucleotide useful fortargeting DMD (e.g., for exon skipping) is 30 nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 409 and 2112-2121.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 45. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 45. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 2097, 2102, 2103, and2122-2146. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 2097, 2102, 2103, and 2122-2146.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon45. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 2097, 2102, 2103, and 2122-2146.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146. In someembodiments, an oligonucleotide useful for targeting DMD (e.g., for exonskipping) is 20-30 (e.g., 20, 25, 30) nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146. Insome embodiments, an oligonucleotide useful for targeting DMD (e.g., forexon skipping) is 20 nucleotides in length, and comprises a region ofcomplementarity to a target sequence comprising at least 4 (e.g., 4, 5,6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one ofSEQ ID NOs: 2097, 2102, 2103, and 2122-2146. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:2097, 2102, 2103, and 2122-2146.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 46. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 46. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 2096 and 2147-2158. In someembodiments, the oligonucleotide comprises a region of complementarityto a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:2096 and 2147-2158.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon46. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 2096 and 2147-2158.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 2096 and 2147-2158. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 2096 and 2147-2158. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:2096 and 2147-2158. In some embodiments, an oligonucleotide useful fortargeting DMD (e.g., for exon skipping) is 30 nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 2096 and 2147-2158.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 50. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 50. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 2096 and 2160-2177. In someembodiments, the oligonucleotide comprises a region of complementarityto a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:2096 and 2160-2177.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon50. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 2096 and 2160-2177.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 2096 and 2160-2177. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 2096 and 2160-2177. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:2096 and 2160-2177. In some embodiments, an oligonucleotide useful fortargeting DMD (e.g., for exon skipping) is 30 nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 2096 and 2160-2177.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 51. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 51. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 402-436. In some embodiments,the oligonucleotide comprises a region of complementarity to a targetsequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutivenucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436. Insome embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 4 (e.g., 4, 5,6, 7, 8) consecutive nucleotides of an ESE as set forth in SEQ ID NO:419.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon51. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 402-436. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of ESEs asset forth in SEQ ID NO: 418 and SEQ ID NO: 419.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 402-436. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 402-436. In some embodiments, an oligonucleotide usefulfor targeting DMD (e.g., for exon skipping) is 20 nucleotides in length,and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 402-436. In someembodiments, an oligonucleotide useful for targeting DMD (e.g., for exonskipping) is 30 nucleotides in length, and comprises a region ofcomplementarity to a target sequence comprising at least 4 (e.g., 4, 5,6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one ofSEQ ID NOs: 402-436.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 20-30 (e.g., 20, 25, 30) nucleotides in length andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in SEQ ID NO: 419. In some embodiments, an oligonucleotideuseful for targeting DMD (e.g., for exon skipping) is 30 nucleotides inlength and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in SEQ ID NO: 419.

In some embodiments, the oligonucleotide is 20-30 (e.g., 20, 25, 30)nucleotides in length and comprises a region of complementarity to atarget sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13,or 14) nucleotides of ESEs as set forth in SEQ ID NO: 418 and SEQ ID NO:419. In some embodiments, the oligonucleotide is 30 nucleotides inlength and comprises a region of complementarity to a target sequencecomprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14)nucleotides of ESEs as set forth in SEQ ID NO: 418 and SEQ ID NO: 419.

Non-limiting examples of oligonucleotides that are useful for DMD exon51 skipping and their target sequences are provided in SEQ ID NOs:437-1241 and SEQ ID NOs: 1242-2046, respectively. In some embodiments,the oligonucleotide is 20-30 nucleotides in length and comprises aregion of complementarity to a target sequence comprising at least 20consecutive nucleotides of any one of SEQ ID NOs: 1242-2046. In someembodiments, the oligonucleotide is 20-30 nucleotides in length andcomprises at least 20 consecutive nucleotides of any one of SEQ ID NOs:437-1241. In some embodiments, the oligonucleotide comprises thenucleotide sequence of any one of SEQ ID NOs: 437-1241. In someembodiments, the oligonucleotide is at least 30 nucleotides (e.g., 30,31, 32, 33, 34, or 35) in length and comprises the nucleotide sequenceof any one of SEQ ID NOs: 437-1241.

In some embodiments, the oligonucleotide is 20-30 nucleotides in lengthand comprises a region of complementarity to a target sequencecomprising at least 20 consecutive nucleotides of any one of SEQ ID NOs:1548, 1550, 1551, 1552, 1555, 1558, 1559, 1562, 1565, 1569, 1577, 1583,1589, 1595, 1600, 1606, 1610, 1614, 1621, 1626, 1629, 1632, 1637, 1640,1643, 1646, 1650, 1655, 1658, and 1662. In some embodiments, theoligonucleotide is 20-30 nucleotides in length and comprises at least 20consecutive nucleotides of any one of SEQ ID NO: 743, 745, 746, 747,750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809,816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853, and 857. In someembodiments, the oligonucleotide comprises the nucleotide sequence ofany one of SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764,772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835,838, 841, 845, 850, 853, and 857. In some embodiments, theoligonucleotide is 30 nucleotides in length and comprises the nucleotidesequence of any one of SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754,757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824,827, 832, 835, 838, 841, 845, 850, 853, and 857.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 52. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 52. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 432 and 2178-2192. In someembodiments, the oligonucleotide comprises a region of complementarityto a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:432 and 2178-2192.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon52. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 432 and 2178-2192.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 432 and 2178-2192. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is20-30 (e.g., 20, 25, 30) nucleotides in length, and comprises a regionof complementarity to a target sequence comprising at least 4 (e.g., 4,5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any oneof SEQ ID NOs: 432 and 2178-2192. In some embodiments, anoligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20nucleotides in length, and comprises a region of complementarity to atarget sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8)consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs:432 and 2178-2192. In some embodiments, an oligonucleotide useful fortargeting DMD (e.g., for exon skipping) is 30 nucleotides in length, andcomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE asset forth in any one of SEQ ID NOs: 432 and 2178-2192.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 53. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 53. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 416, 430, 431, 2108, 2114,2127, and 2193-2213. In some embodiments, the oligonucleotide comprisesa region of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and2193-2213.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon53. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and2193-2213. In some embodiments, an oligonucleotide useful for targetingDMD (e.g., for exon skipping) is 20-30 (e.g., 20, 25, 30) nucleotides inlength, and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108,2114, 2127, and 2193-2213. In some embodiments, an oligonucleotideuseful for targeting DMD (e.g., for exon skipping) is 20 nucleotides inlength, and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108,2114, 2127, and 2193-2213. In some embodiments, an oligonucleotideuseful for targeting DMD (e.g., for exon skipping) is 30 nucleotides inlength, and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108,2114, 2127, and 2193-2213.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising one or more full orpartial ESEs of DMD exon 55. In some embodiments, the oligonucleotidecomprises a region of complementarity to a target sequence comprising atleast 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE ofDMD exon 55. In some embodiments, the oligonucleotide comprises a regionof complementarity to a target sequence comprising one or more full orpartial ESEs as set forth in SEQ ID NOs: 2097, 2102, 2103, 2116, 2147,2199, and 2214-2238. In some embodiments, the oligonucleotide comprisesa region of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and2214-2238.

In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon55. In some embodiments, the oligonucleotide comprises a region ofcomplementarity to a target sequence comprising at least 6 (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotidesof one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forthin SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238.

In some embodiments, an oligonucleotide useful for targeting DMD (e.g.,for exon skipping) is 18-35 nucleotides in length, and comprises aregion of complementarity to a target sequence comprising at least 4(e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forthin any one of SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and2214-2238. In some embodiments, an oligonucleotide useful for targetingDMD (e.g., for exon skipping) is 20-30 (e.g., 20, 25, 30) nucleotides inlength, and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, 2116,2147, 2199, and 2214-2238. In some embodiments, an oligonucleotideuseful for targeting DMD (e.g., for exon skipping) is 20 nucleotides inlength, and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, 2116,2147, 2199, and 2214-2238. In some embodiments, an oligonucleotideuseful for targeting DMD (e.g., for exon skipping) is 30 nucleotides inlength, and comprises a region of complementarity to a target sequencecomprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotidesof an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, 2116,2147, 2199, and 2214-2238.

In some embodiments, any one of the oligonucleotides useful fortargeting DMD (e.g., for exon skipping) is a phosphorodiamidatemorpholino oligomer (PMO).

Additional examples of oligonucleotides targeting DMD (e.g., for exonskipping) are provided in U.S. Patent Application Publication2013-072541, published Mar. 21, 2013, entitled “ADENO-ASSOCIATED VIRALVECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSIBLE-DOMAINPROTEIN”; U.S. Patent Application Publication 2015-191725, publishedJul. 9, 2015, entitled “OLIGONUCLEOTIDE FOR THE TREATMENT OF MUSCULARDYSTROPHY PATIENTS”; U.S. Patent Application Publication 2015-196670,published Jul. 16, 2015, entitled “COMPOSITIONS AND METHODS FOR DUCHENNEMUSCULAR DYSTROPHY GENE THERAPY”; U.S. Patent Application Publication2017-349905, published Dec. 7, 2017, entitled “GENOME EDITING WITH SPLITCAS9 EXPRESSED FROM TWO VECTORS”; U.S. Patent Application Publication2018-028554, published Feb. 1, 2018, entitled “OLIGOMERS HAVING BICYCLICSCAFFOLD MOEITIES”; U.S. Patent Application Publication 2018-171333,published Jun. 21, 2018, entitled “ANTISENSE MOLECULES AND METHODS FORTREATING PATHOLOGIES”; U.S. Patent Application Publication 2018-179538,published Jun. 28, 2018, entitled “ANTISENSE NUCLEIC ACIDS”; U.S. PatentApplication Publication 2018-265859, published Sep. 20, 2018, entitled“MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF”; U.S. PatentApplication Publication 2018-369400, published Dec. 27, 2018, entitled“NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXONSKIPPING”; U.S. Patent Application Publication 2019-000986, publishedJan. 3, 2019, entitled “NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS ANDMETHODS OF INDUCING EXON SKIPPING”; U.S. Patent Application Publication2019-008986, published Jan. 10, 2019, entitled “OLIGONUCLEOTIDECOMPOSITIONS AND METHODS THEREOF”; U.S. Patent Application Publication2019-112604, published Apr. 18, 2019, entitled “METHODS AND MEANS FOREFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA”;U.S. Patent Application Publication 2019-119679, published Apr. 25,2019, entitled “METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 INDUCHENNE MUSCULAR DYSTROPHY PRE-MRNA”; U.S. Patent ApplicationPublication 2019-127733, published May 2, 2019, entitled“OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF”; U.S. PatentApplication Publication 2019-151476, published May 23, 2019, entitled“THERAPEUTIC APPLICATIONS OF CPF1-BASED GENOME EDITING”; U.S. PatentApplication Publication 2019-177723, published Jun. 13, 2019, entitled“COMPOSITIONS AND METHODS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY ANDRELATED DISORDERS”; U.S. Patent Application Publication 2019-177725,published Jun. 13, 2019, entitled “METHODS AND MEANS FOR EFFICIENTSKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA”; U.S.Patent Application Publication 2019-209604, published Jul. 11, 2019,entitled “OLIGONUCLEOTIDES, COMPOSITIONS AND METHODS THEREOF”; U.S.Patent Application Publication 2019-249173, published Aug. 15, 2019,entitled “METHODS AND COMPOSITIONS OF BIOLOGICALLY ACTIVE AGENTS”; U.S.Patent Application Publication 2019-270994, published Sep. 5, 2019,entitled “ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES”;U.S. Patent Application Publication 2019-284556, published Sep. 19,2019, entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD”; U.S.Patent Application Publication 2019-323010, published Oct. 24, 2019,entitled “ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING ANDMETHODS OF USE THEREOF”; U.S. Patent Application Publication2019-330626, published Oct. 31, 2019, entitled “COMPOUNDS AND METHODSFOR USE IN DYSTROPHIN TRANSCRIPT”; U.S. Patent Application Publication2019-338311, published Nov. 7, 2019, entitled “OPTIMIZED STRATEGY FOREXON SKIPPING MODIFICATIONS USING CRISPR/CAS9 WITH TRIPLE GUIDESEQUENCES”; U.S. Patent Application Publication 2019-359982, publishedNov. 28, 2019, entitled “COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY”;U.S. Patent Application Publication 2019-364862, published Dec. 5, 2019,entitled “DMD REPORTER MODELS CONTAINING HUMANIZED DUCHENNE MUSCULARDYSTROPHY MUTATIONS”; U.S. Patent Application Publication 2019-390197,published Dec. 26, 2019, entitled “OLIGONUCLEOTIDE COMPOSITIONS ANDMETHODS THEREOF”; U.S. Patent Application Publication 2020-040337,published Feb. 6, 2020, entitled “COMPOSITIONS FOR TREATING MUSCULARDYSTROPHY”; U.S. Pat. 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No. 10,876,114, issued Dec. 29,2020, entitled “METHODS AND MEANS FOR EFFICIENT SKIPPING OF AT LEAST ONEOF THE FOLLOWING EXONS OF THE HUMAN DUCHENNE MUSCULAR DYSTROPHY GENE:43, 46, 50-53”; U.S. Pat. No. 6,100,099, issued Aug. 8, 2000, entitled“TEST STRIP HAVING A DIAGONAL ARRAY OF CAPTURE SPOTS”; U.S. Pat. No.6,210,898, issued Apr. 3, 2001, entitled “METHOD OF PERFORMINGIMMUNOCHROMATOGRAPHY”; U.S. Pat. No. 7,973,015, issued Jul. 5, 2011,entitled “INDUCTION OF EXON SKIPPING IN EUKARYOTIC CELLS”; U.S. Pat. No.8,039,608, issued Oct. 18, 2011, entitled “BIOINFORMATICALLY DETECTABLEGROUP OF NOVEL REGULATORY GENES AND USES THEREOF”; U.S. Pat. No.8,361,979, issued Jan. 29, 2013, entitled “MEANS AND METHOD FOR INDUCINGEXON-SKIPPING”; U.S. Pat. No. 8,802,437, issued Aug. 12, 2014, entitled“MEGANUCLEASE REAGENTS OF USES THEREOF FOR TREATING GENETIC DISEASESCAUSED BY FRAME SHIFT/NON SENSE MUTATIONS”; U.S. Pat. No. 8,865,883,issued Oct. 21, 2014, entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FORDMD”; U.S. Pat. No. 9,657,049, issued May 23, 2017, entitled “ENANUCLEIC ACID PHARMACEUTICALS CAPABLE OF MODIFYING SPLICING OF MRNAPRECURSORS”; U.S. Pat. No. 9,657,050, issued May 23, 2017, entitled “ENANUCLEIC ACID PHARMACEUTICALS CAPABLE OF MODIFYING SPLICING OF MRNAPRECURSORS”; U.S. Pat. No. 9,988,629, issued Jun. 5, 2018, entitled“ANTISENSE NUCLEIC ACIDS”; International Patent Publication WO2011/078797 A2, published Jun. 30, 2011, entitled “ANTISENSEOLIGONUCLEOTIDES AND USES THEREOF”; International Patent Publication WO2011/154427 A1, published Dec. 15, 2011, entitled “MODIFIED SNRNAS FORUSE IN THERAPY”; International Patent Publication WO 2018/007475 A1,published Jan. 11, 2018, entitled “PRE-MRNA SPLICE SWITCHING ORMODULATING OLIGONUCLEOTIDES COMPRISING BICYCLIC SCAFFOLD MOIETIES, WITHIMPROVED CHARACTERISTICS FOR THE TREATMENT OF GENETIC DISORDERS”;International Patent Publication WO 2018/014042 A1, published Jan. 18,2018, entitled “COMPOUNDS AND METHODS FOR MODULATION OF DYSTROPHINTRANSCRIPT”; International Patent Publication WO 2018/017754 A1,published Jan. 25, 2018, entitled “THERAPEUTIC APPLICATIONS OFCPF1-BASED GENOME EDITING”; International Patent Publication WO2018/107003 A1, published Jun. 14, 2018, entitled “DMD REPORTER MODELSCONTAINING HUMANIZED DUCHENNE MUSCULAR DYSTROPHY MUTATIONS”;International Patent Publication WO 2018/129296 A1, published Jul. 12,2018, entitled “OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USINGCRISPR/CAS9 WITH TRIPLE GUIDE SEQUENCES”; International PatentPublication WO 2019/014772 A1, published Jan. 24, 2019, entitled“ANTISENSE OLIGONUCLEOTIDES THAT BIND TO EXON 51 OF HUMAN DYSTROPHINPRE-MRNA”; International Patent Publication WO 2019/059973 A1, publishedMar. 28, 2019, entitled “EXON SKIPPING OLIGOMER CONJUGATES FOR MUSCULARDYSTROPHY”; International Patent Publication WO 2019/060775 A1,published Mar. 28, 2019, entitled “NUCLEIC ACID-POLYPEPTIDE COMPOSITIONSAND METHODS OF INDUCING EXON SKIPPING”; International Patent PublicationWO 2019/067975 A1, published Apr. 4, 2019, entitled “COMBINATIONTHERAPIES FOR TREATING MUSCULAR DYSTROPHY”; International PatentPublication WO 2019/092507 A2, published May 16, 2019, entitled“CRISPR/CAS SYSTEMS FOR TREATMENT OF DMD”; International PatentPublication WO 2019/136216 A1, published Jul. 11, 2019, entitled“THERAPEUTIC CRISPR/CAS9 COMPOSITIONS AND METHODS OF USE”; InternationalPatent Publication WO 2019/152609 A1, published Aug. 8, 2019, entitled“COMPOSITIONS AND METHODS FOR CORRECTING DYSTROPHIN MUTATIONS IN HUMANCARDIOMYOCYTES”; International Patent Publication WO 2019/200185 A1,published Oct. 17, 2019, entitled “OLIGONUCLEOTIDE COMPOSITIONS ANDMETHODS OF USE THEREOF”; International Patent Publication WO 2019/215333A1, published Nov. 14, 2019, entitled “OLIGONUCLEOTIDES CONJUGATESCOMPRISING 7′-5′-ALPHA-ANOMERIC-BICYCLIC SUGAR NUCLEOSIDES”;International Patent Publication WO 2019/241385 A2, published Dec. 19,2019, entitled “EXON SKIPPING OLIGOMERS FOR MUSCULAR DYSTROPHY”;International Patent Publication WO 2019/246480 A1, published Dec. 26,2019, entitled “CORRECTION OF DYSTROPHIN EXON 43, EXON 45, OR EXON 52DELETIONS IN DUCHENNE MUSCULAR DYSTROPHY”; International PatentPublication WO 2020/028832 A1, published Feb. 6, 2020, entitled “MUSCLETARGETING COMPLEXES AND USES THEREOF FOR TREATING DYSTROPHINOPATHIES”;International Patent Publication WO 2018/091544 A1, published May 24,2018, entitled “SUBSTANCES FOR TARGETING VARIOUS SELECTED ORGANS ORTISSUES”; International Patent Publication WO 2018/098480 A1, publishedMay 31, 2018, entitled “PREVENTION OF MUSCULAR DYSTROPHY BYCRISPR/CPF1-MEDIATED GENE EDITING”; International Patent Publication WO1993/020227 A1, published Oct. 14, 1993, entitled “METHOD OF MULTIPLEXLIGASE CHAIN REACTION”; International Patent Publication WO 2013/100190A1, published Jul. 4, 2013, entitled “ANTISENSE NUCLEIC ACID”;International Patent Publication WO 2013/163628 A2, published Oct. 31,2013, entitled “GENETIC CORRECTION OF MUTATED GENES”; InternationalPatent Publication WO 2007/135105 A1, published Nov. 29, 2007, entitled“MEANS AND METHOD FOR INDUCING EXON-SKIPPING”; International PatentPublication WO 2011/150408 A2, published Dec. 1, 2011, entitled“OLIGONUCLEOTIDE ANALOGUES HAVING MODIFIED INTERSUBUNIT LINKAGES AND/ORTERMINAL GROUPS”; International Patent Publication WO 2012/029986 A1,published Mar. 8, 2012, entitled “ANTISENSE NUCLEIC ACID”; the contentsof each of which are incorporated herein in their entireties.

Examples of oligonucleotides for promoting DMD gene editing includeInternational Patent Publication WO2018053632A1, published Mar. 29,2018, entitled “METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORINGDYSTROPHIN EXPRESSION AND USES THEREOF”; International PatentPublication WO2017049407A1, published Mar. 30, 2017, entitled“MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF”; InternationalPatent Publication WO2016161380A1, published Oct. 6, 2016, entitled“CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNEMUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY”; International PatentPublication WO2017095967, published Jun. 8, 2017, entitled “THERAPEUTICTARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITINGAND METHODS OF USE”; International Patent Publication WO2017072590A1,published May 4, 2017, entitled “MATERIALS AND METHODS FOR TREATMENT OFDUCHENNE MUSCULAR DYSTROPHY”; International Patent PublicationWO2018098480A1, published May 31, 2018, entitled “PREVENTION OF MUSCULARDYSTROPHY BY CRISPR/CPF1-MEDIATED GENE EDITING”; US Patent ApplicationPublication US20170266320A1, published Sep. 21, 2017, entitled“RNA-Guided Systems for In Vivo Gene Editing”; International PatentPublication WO2016025469A1, published Feb. 18, 2016, entitled“PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR/CAS9-MEDIATED GENE EDITING”;U.S. Patent Application Publication 2016/0201089, published Jul. 14,2016, entitled “RNA-GUIDED GENE EDITING AND GENE REGULATION”; and U.S.Patent Application Publication 2013/0145487, published Jun. 6, 2013,entitled “MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THEDYSTROPHIN GENE AND USES THEREOF”, the contents of each of which areincorporated herein in their entireties. In some embodiments, anoligonucleotide may have a region of complementarity to DMD genesequences of multiple species, e.g., selected from human, mouse andnon-human species.

In some embodiments, the oligonucleotide may have region ofcomplementarity to a mutant DMD allele, for example, a DMD allele withat least one mutation in any of exons 1-79 of DMD in humans that leadsto a frameshift and improper RNA splicing/processing.

In some embodiments, the oligonucleotide may target lncRNA or mRNA,e.g., for degradation. In some embodiments, the oligonucleotide maytarget, e.g., for degradation, a nucleic acid encoding a proteininvolved in a mismatch repair pathway, e.g., MSH2, MutLalpha, MutSbeta,MutLalpha. Non-limiting examples of proteins involved in mismatch repairpathways, for which mRNAs encoding such proteins may be targeted byoligonucleotides described herein, are described in Iyer, R. R. et al.,“DNA triplet repeat expansion and mismatch repair” Annu Rev Biochem.2015; 84:199-226.; and Schmidt M. H. and Pearson C. E.,“Disease-associated repeat instability and mismatch repair” DNA Repair(Amst). 2016 February; 38:117-26.

In some embodiments, any one of the oligonucleotides can be in saltform, e.g., as sodium, potassium, or magnesium salts.

In some embodiments, the 5′ or 3′ nucleoside (e.g., terminal nucleoside)of any one of the oligonucleotides described herein is conjugated to anamine group, optionally via a spacer. In some embodiments, the spacercomprises an aliphatic moiety. In some embodiments, the spacer comprisesa polyethylene glycol moiety. In some embodiments, a phosphodiesterlinkage is present between the spacer and the 5′ or 3′ nucleoside of theoligonucleotide. In some embodiments, the 5′ or 3′ nucleoside (e.g.,terminal nucleoside) of any of the oligonucleotides described herein isconjugated to a spacer that is a substituted or unsubstituted aliphatic,substituted or unsubstituted heteroaliphatic, substituted orunsubstituted carbocyclylene, substituted or unsubstitutedheterocyclylene, substituted or unsubstituted arylene, substituted orunsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—,—C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—,—NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—,—OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combinationthereof; each R^(A) is independently hydrogen or substituted orunsubstituted alkyl. In certain embodiments, the spacer is a substitutedor unsubstituted alkylene, substituted or unsubstituted heterocyclylene,substituted or unsubstituted heteroarylene, —O—, —N(R^(A))—, or—C(═O)N(R^(A))₂, or a combination thereof.

In some embodiments, the 5′ or 3′ nucleoside of any one of theoligonucleotides described herein is conjugated to a compound of theformula —NH₂—(CH₂)_(n)—, wherein n is an integer from 1 to 12. In someembodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, aphosphodiester linkage is present between the compound of the formulaNH₂—(CH₂)_(n)— and the 5′ or 3′ nucleoside of the oligonucleotide. Insome embodiments, a compound of the formula NH₂—(CH₂)₆— is conjugated tothe oligonucleotide via a reaction between 6-amino-1-hexanol(NH₂—(CH₂)₆—OH) and the 5′ phosphate of the oligonucleotide.

In some embodiments, the oligonucleotide is conjugated to a targetingagent, e.g., a muscle targeting agent such as an anti-TfR antibody,e.g., via the amine group.

a. Oligonucleotide Size/Sequence

Oligonucleotides may be of a variety of different lengths, e.g.,depending on the format. In some embodiments, an oligonucleotide is 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, the oligonucleotide is 8 to 50 nucleotides inlength, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 21 to 23 nucleotides in lengths, etc.

In some embodiments, a complementary nucleic acid sequence of anoligonucleotide for purposes of the present disclosure is specificallyhybridizable or specific for the target nucleic acid when binding of thesequence to the target molecule (e.g., mRNA) interferes with thefunction of the target (e.g., mRNA) to cause a change of activity (e.g.,inhibiting translation, altering splicing, exon skipping) or expression(e.g., degrading a target mRNA) and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the sequence tonon-target sequences under conditions in which avoidance of non-specificbinding is desired, e.g., under physiological conditions in the case ofin vivo assays or therapeutic treatment, and in the case of in vitroassays, under conditions in which the assays are performed undersuitable conditions of stringency. Thus, in some embodiments, anoligonucleotide may be at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100%complementary to the consecutive nucleotides of a target nucleic acid.In some embodiments a complementary nucleotide sequence need not be 100%complementary to that of its target to be specifically hybridizable orspecific for a target nucleic acid.

In some embodiments, an oligonucleotide comprises region ofcomplementarity to a target nucleic acid that is in the range of 8 to15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides inlength. In some embodiments, a region of complementarity of anoligonucleotide to a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 nucleotides in length. In some embodiments, the region ofcomplementarity is complementary with at least 8 consecutive nucleotidesof a target nucleic acid. In some embodiments, an oligonucleotide maycontain 1, 2 or 3 base mismatches compared to the portion of theconsecutive nucleotides of target nucleic acid. In some embodiments theoligonucleotide may have up to 3 mismatches over 15 bases, or up to 2mismatches over 10 bases.

In some embodiments, the oligonucleotide is complementary (e.g., atleast 85% at least 90%, at least 95%, or 100%) to a target sequence ofthe any one of the oligonucleotides described herein (e.g., theoligonucleotides listed in Table 14). In some embodiments, theoligonucleotide is complementary (e.g., at least 85% at least 90%, atleast 95%, or 100%) to a target sequence of the any one of theoligonucleotides provided by SEQ ID NO: 437-1241. In some embodiments,such target sequence is 100% complementary to an oligonucleotide listedin Table 14. In some embodiments, such target sequence is 100%complementary to an oligonucleotide provided by SEQ ID NO: 437-1241. Insome embodiments, the oligonucleotide is complementary (e.g., at least85% at least 90%, at least 95%, or 100%) to a target sequence providedherein (e.g., a target sequence of any one of the oligonucleotideslisted in Table 14). In some embodiments, the oligonucleotide iscomplementary (e.g., at least 85% at least 90%, at least 95%, or 100%)to a target sequence of any one of the oligonucleotides provided by SEQID NO: 1242-2046.

In some embodiments, any one or more of the thymine bases (T's) in anyone of the oligonucleotides provided herein (e.g., the oligonucleotideslisted in Table 14) may optionally be uracil bases (U's), and/or any oneor more of the U's in the oligonucleotides provided herein mayoptionally be T's. In some embodiments, any one or more of the thyminebases (T's) in any one of the oligonucleotides provided by SEQ ID NOs:437-1241 or in an oligonucleotide complementary to any one of SEQ IDNOs: 1242-2046 may optionally be uracil bases (U's), and/or any one ormore of the U's in the oligonucleotides may optionally be T's.

b. Oligonucleotide Modifications:

The oligonucleotides described herein may be modified, e.g., comprise amodified sugar moiety, a modified internucleoside linkage, a modifiednucleotide and/or (e.g., and) combinations thereof. In addition, in someembodiments, oligonucleotides may exhibit one or more of the followingproperties: do not mediate alternative splicing; are not immunestimulatory; are nuclease resistant; have improved cell uptake comparedto unmodified oligonucleotides; are not toxic to cells or mammals; haveimproved endosomal exit internally in a cell; minimizes TLR stimulation;or avoid pattern recognition receptors. Any of the modified chemistriesor formats of oligonucleotides described herein can be combined witheach other. For example, one, two, three, four, five, or more differenttypes of modifications can be included within the same oligonucleotide.

In some embodiments, certain nucleotide modifications may be used thatmake an oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide oroligoribonucleotide molecules; these modified oligonucleotides surviveintact for a longer time than unmodified oligonucleotides. Specificexamples of modified oligonucleotides include those comprising modifiedbackbones, for example, modified internucleoside linkages such asphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Accordingly, oligonucleotides of thedisclosure can be stabilized against nucleolytic degradation such as bythe incorporation of a modification, e.g., a nucleotide modification.

In some embodiments, an oligonucleotide may be of up to 50 or up to 100nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or morenucleotides of the oligonucleotide are modified nucleotides. Theoligonucleotide may be of 8 to 30 nucleotides in length in which 2 to10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to30 nucleotides of the oligonucleotide are modified nucleotides. Theoligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4,2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides.Optionally, the oligonucleotides may have every nucleotide except 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. Oligonucleotidemodifications are described further herein.

c. Modified Nucleosides

In some embodiments, the oligonucleotide described herein comprises atleast one nucleoside modified at the 2′ position of the sugar. In someembodiments, an oligonucleotide comprises at least one 2′-modifiednucleoside. In some embodiments, all of the nucleosides in theoligonucleotide are 2′-modified nucleosides.

In some embodiments, the oligonucleotide described herein comprises oneor more non-bicyclic 2′-modified nucleosides, e.g., 2′-deoxy, 2′-fluoro(2′-F), 2′-O-methyl (2′-0-Me), 2′-O-methoxyethyl (2′-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O-N-methylacetamido (2′-O-NMA) modified nucleoside.

In some embodiments, the oligonucleotide described herein comprises oneor more 2′-4′ bicyclic nucleosides in which the ribose ring comprises abridge moiety connecting two atoms in the ring, e.g., connecting the2′-O atom to the 4′-C atom via a methylene (LNA) bridge, an ethylene(ENA) bridge, or a (S)-constrained ethyl (cEt) bridge. Examples of LNAsare described in International Patent Application PublicationWO/2008/043753, published on Apr. 17, 2008, and entitled “RNA AntagonistCompounds For The Modulation Of PCSK9”, the contents of which areincorporated herein by reference in its entirety. Examples of ENAs areprovided in International Patent Publication No. WO 2005/042777,published on May 12, 2005, and entitled “APP/ENA Antisense”; Morita etal., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. GeneTher., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149,2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005;the disclosures of which are incorporated herein by reference in theirentireties. Examples of cEt are provided in U.S. Pat. Nos. 7,101,993;7,399,845 and 7,569,686, each of which is herein incorporated byreference in its entirety.

In some embodiments, the oligonucleotide comprises a modified nucleosidedisclosed in one of the following United States Patent or PatentApplication Publications: U.S. Pat. No. 7,399,845, issued on Jul. 15,2008, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Pat.No. 7,741,457, issued on Jun. 22, 2010, and entitled “6-ModifiedBicyclic Nucleic Acid Analogs”; U.S. Pat. No. 8,022,193, issued on Sep.20, 2011, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S.Pat. No. 7,569,686, issued on Aug. 4, 2009, and entitled “Compounds AndMethods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Pat. No.7,335,765, issued on Feb. 26, 2008, and entitled “Novel Nucleoside AndOligonucleotide Analogues”; U.S. Pat. No. 7,314,923, issued on Jan. 1,2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”;U.S. Pat. No. 7,816,333, issued on Oct. 19, 2010, and entitled“Oligonucleotide Analogues And Methods Utilizing The Same” and USPublication Number 2011/0009471 now U.S. Pat. No. 8,957,201, issued onFeb. 17, 2015, and entitled “Oligonucleotide Analogues And MethodsUtilizing The Same”, the entire contents of each of which areincorporated herein by reference for all purposes.

In some embodiments, the oligonucleotide comprises at least one modifiednucleoside that results in an increase in Tm of the oligonucleotide in arange of 1° C., 2° C., 3° C., 4° C., or 5° C. compared with anoligonucleotide that does not have the at least one modified nucleoside.The oligonucleotide may have a plurality of modified nucleosides thatresult in a total increase in Tm of the oligonucleotide in a range of 2°C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20°C., 25° C., 30° C., 35° C., 40° C., 45° C. or more compared with anoligonucleotide that does not have the modified nucleoside.

The oligonucleotide may comprise a mix of nucleosides of differentkinds. For example, an oligonucleotide may comprise a mix of2′-deoxyribonucleosides or ribonucleosides and 2′-fluoro modifiednucleosides. An oligonucleotide may comprise a mix ofdeoxyribonucleosides or ribonucleosides and 2′-O-Me modifiednucleosides. An oligonucleotide may comprise a mix of 2′-fluoro modifiednucleosides and 2′-O-Me modified nucleosides. An oligonucleotide maycomprise a mix of 2′-4′ bicyclic nucleosides and 2′-MOE, 2′-fluoro, or2′-O-Me modified nucleosides. An oligonucleotide may comprise a mix ofnon-bicyclic 2′-modified nucleosides (e.g., 2′-MOE, 2′-fluoro, or2′-O-Me) and 2′-4′ bicyclic nucleosides (e.g., LNA, ENA, cEt).

The oligonucleotide may comprise alternating nucleosides of differentkinds. For example, an oligonucleotide may comprise alternating2′-deoxyribonucleosides or ribonucleosides and 2′-fluoro modifiednucleosides. An oligonucleotide may comprise alternatingdeoxyribonucleosides or ribonucleosides and 2′-O-Me modifiednucleosides. An oligonucleotide may comprise alternating 2′-fluoromodified nucleosides and 2′-O-Me modified nucleosides. Anoligonucleotide may comprise alternating 2′-4′ bicyclic nucleosides and2′-MOE, 2′-fluoro, or 2′-O-Me modified nucleosides. An oligonucleotidemay comprise alternating non-bicyclic 2′-modified nucleosides (e.g.,2′-MOE, 2′-fluoro, or 2′-O-Me) and 2′-4′ bicyclic nucleosides (e.g.,LNA, ENA, cEt).

In some embodiments, an oligonucleotide described herein comprises a5′-vinylphosphonate modification, one or more abasic residues, and/orone or more inverted abasic residues.

d. Internucleoside Linkages/Backbones

In some embodiments, oligonucleotide may contain a phosphorothioate orother modified internucleoside linkage. In some embodiments, theoligonucleotide comprises phosphorothioate internucleoside linkages. Insome embodiments, the oligonucleotide comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the oligonucleotide comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, oligonucleotides comprise modified internucleoside linkagesat the first, second, and/or (e.g., and) third internucleoside linkageat the 5′ or 3′ end of the nucleotide sequence.

Phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

In some embodiments, oligonucleotides may have heteroatom backbones,such as methylene(methylimino) or MMI backbones; amide backbones (see DeMesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones(see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleicacid (PNA) backbones (wherein the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleotidesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

e. Stereospecific Oligonucleotides

In some embodiments, internucleotidic phosphorus atoms ofoligonucleotides are chiral, and the properties of the oligonucleotidesby adjusted based on the configuration of the chiral phosphorus atoms.In some embodiments, appropriate methods may be used to synthesizeP-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., asdescribed in Oka N, Wada T, Stereocontrolled synthesis ofoligonucleotide analogs containing chiral internucleotidic phosphorusatoms. Chem Soc Rev. 2011 December; 40(12):5829-43.) In someembodiments, phosphorothioate containing oligonucleotides comprisenucleoside units that are joined together by either substantially all Spor substantially all Rp phosphorothioate intersugar linkages areprovided. In some embodiments, such phosphorothioate oligonucleotideshaving substantially chirally pure intersugar linkages are prepared byenzymatic or chemical synthesis, as described, for example, in U.S. Pat.No. 5,587,261, issued on Dec. 12, 1996, the contents of which areincorporated herein by reference in their entirety. In some embodiments,chirally controlled oligonucleotides provide selective cleavage patternsof a target nucleic acid. For example, in some embodiments, a chirallycontrolled oligonucleotide provides single site cleavage within acomplementary sequence of a nucleic acid, as described, for example, inUS Patent Application Publication 20170037399 A1, published on Feb. 2,2017, entitled “CHIRAL DESIGN”, the contents of which are incorporatedherein by reference in their entirety.

f. Morpholinos

In some embodiments, the oligonucleotide may be a morpholino-basedcompounds. Morpholino-based oligomeric compounds are described in DwaineA. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510);Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243,209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra etal., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No.5,034,506, issued Jul. 23, 1991. In some embodiments, themorpholino-based oligomeric compound is a phosphorodiamidate morpholinooligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther.,3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; thedisclosures of which are incorporated herein by reference in theirentireties).

g. Peptide Nucleic Acids (PNAs)

In some embodiments, both a sugar and an internucleoside linkage (thebackbone) of the nucleotide units of an oligonucleotide are replacedwith novel groups. In some embodiments, the base units are maintainedfor hybridization with an appropriate nucleic acid target compound. Onesuch oligomeric compound, an oligonucleotide mimetic that has been shownto have excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, forexample, an aminoethylglycine backbone. The nucleobases are retained andare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative publication that report thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

h. Gapmers

In some embodiments, an oligonucleotide described herein is a gapmer. Agapmer oligonucleotide generally has the formula 5′-X-Y-Z-3′, with X andZ as flanking regions around a gap region Y. In some embodiments,flanking region X of formula 5′-X-Y-Z-3′ is also referred to as Xregion, flanking sequence X, 5′ wing region X, or 5′ wing segment. Insome embodiments, flanking region Z of formula 5′-X-Y-Z-3′ is alsoreferred to as Z region, flanking sequence Z, 3′ wing region Z, or 3′wing segment. In some embodiments, gap region Y of formula 5′-X-Y-Z-3′is also referred to as Y region, Y segment, or gap-segment Y. In someembodiments, each nucleoside in the gap region Y is a2′-deoxyribonucleoside, and neither the 5′ wing region X or the 3′ wingregion Z contains any 2′-deoxyribonucleosides.

In some embodiments, the Y region is a contiguous stretch ofnucleotides, e.g., a region of 6 or more DNA nucleotides, which arecapable of recruiting an RNAse, such as RNAse H. In some embodiments,the gapmer binds to the target nucleic acid, at which point an RNAse isrecruited and can then cleave the target nucleic acid. In someembodiments, the Y region is flanked both 5′ and 3′ by regions X and Zcomprising high-affinity modified nucleosides, e.g., one to sixhigh-affinity modified nucleosides. Examples of high affinity modifiednucleosides include, but are not limited to, 2′-modified nucleosides(e.g., 2′-MOE, 2′O-Me, 2′-F) or 2′-4′ bicyclic nucleosides (e.g., LNA,cEt, ENA). In some embodiments, the flanking sequences X and Z may be of1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. Theflanking sequences X and Z may be of similar length or of dissimilarlengths. In some embodiments, the gap-segment Y may be a nucleotidesequence of 5-20 nucleotides, 5-15 twelve nucleotides, or 6-10nucleotides in length.

In some embodiments, the gap region of the gapmer oligonucleotides maycontain modified nucleotides known to be acceptable for efficient RNaseH action in addition to DNA nucleotides, such as C4′-substitutednucleotides, acyclic nucleotides, and arabino-configured nucleotides. Insome embodiments, the gap region comprises one or more unmodifiedinternucleosides. In some embodiments, one or both flanking regions eachindependently comprise one or more phosphorothioate internucleosidelinkages (e.g., phosphorothioate internucleoside linkages or otherlinkages) between at least two, at least three, at least four, at leastfive or more nucleotides. In some embodiments, the gap region and twoflanking regions each independently comprise modified internucleosidelinkages (e.g., phosphorothioate internucleoside linkages or otherlinkages) between at least two, at least three, at least four, at leastfive or more nucleotides.

A gapmer may be produced using appropriate methods. Representative U.S.patents, U.S. patent publications, and PCT publications that teach thepreparation of gapmers include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922;5,898,031; 7,015,315; 7,101,993; 7,399,845; 7,432,250; 7,569,686;7,683,036; 7,750,131; 8,580,756; 9,045,754; 9,428,534; 9,695,418;10,017,764; 10,260,069; 9,428,534; 8,580,756; U.S. patent publicationNos. US20050074801, US20090221685; US20090286969, US20100197762, andUS20110112170; PCT publication Nos. WO2004069991; WO2005023825;WO2008049085 and WO2009090182; and EP Patent No. EP2,149,605, each ofwhich is herein incorporated by reference in its entirety.

In some embodiments, a gapmer is 10-40 nucleosides in length. Forexample, a gapmer may be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15,15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40,25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length. In someembodiments, a gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,or 40 nucleosides in length.

In some embodiments, the gap region Y in a gapmer is 5-20 nucleosides inlength. For example, the gap region Y may be 5-20, 5-15, 5-10, 10-20,10-15, or 15-20 nucleosides in length. In some embodiments, the gapregion Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleosides in length. In some embodiments, each nucleoside in the gapregion Y is a 2′-deoxyribonucleoside. In some embodiments, allnucleosides in the gap region Y are 2′-deoxyribonucleosides. In someembodiments, one or more of the nucleosides in the gap region Y is amodified nucleoside (e.g., a 2′ modified nucleoside such as thosedescribed herein). In some embodiments, one or more cytosines in the gapregion Y are optionally 5-methyl-cytosines. In some embodiments, eachcytosine in the gap region Y is a 5-methyl-cytosines.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X-Y-Z-3′formula) and the 3′wing region of a gapmer (Z in the 5′-X-Y-Z-3′formula) are independently 1-20 nucleosides long. For example, the5′wing region of a gapmer (X in the 5′-X-Y-Z-3′ formula) and the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) may be independently1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 1-2, 2-5, 2-7, 3-5, 3-7, 5-20, 5-15,5-10, 10-20, 10-15, or 15-20 nucleosides long. In some embodiments, the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) areindependently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 nucleosides long. In some embodiments, the 5′wing regionof the gapmer (X in the 5′-X-Y-Z-3′ formula) and the 3′wing region ofthe gapmer (Z in the 5′-X-Y-Z-3′ formula) are of the same length. Insome embodiments, the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) and the 3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′formula) are of different lengths. In some embodiments, the 5′wingregion of the gapmer (X in the 5′-X-Y-Z-3′ formula) is longer than the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula). In someembodiments, the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) is shorter than the 3′wing region of the gapmer (Z in the5′-X-Y-Z-3′ formula).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ of 5-10-5, 4-12-4,3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3,2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1,2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2,1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3,1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2,1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1,2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3,1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2,2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5,5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4,4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1,2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1,1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4,4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7,7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2,3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1,2-17-2, 1-16-4, 4-16-1, 2-16-3, 3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3,1-14-6, 6-14-1, 2-14-5, 5-14-2, 3-14-4, 4-14-3, 1-13-7, 7-13-1, 2-13-6,6-13-2, 3-13-5, 5-13-3, 4-13-4, 1-12-8, 8-12-1, 2-12-7, 7-12-2, 3-12-6,6-12-3, 4-12-5, 5-12-4, 2-11-8, 8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4,5-11-5, 1-20-1, 1-19-2, 2-19-1, 1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1,2-17-3, 3-17-2, 1-16-5, 2-16-4, 4-16-2, 3-16-3, 1-15-6, 6-15-1, 2-15-5,5-15-2, 3-15-4, 4-15-3, 1-14-7, 7-14-1, 2-14-6, 6-14-2, 3-14-5, 5-14-3,4-14-4, 1-13-8, 8-13-1, 2-13-7, 7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4,2-12-8, 8-12-2, 3-12-7, 7-12-3, 4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3,4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1,2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3,1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6,6-15-2, 3-15-5, 5-15-3, 4-15-4, 1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6,6-14-3, 4-14-5, 5-14-4, 2-13-8, 8-13-2, 3-13-7, 7-13-3, 4-13-6, 6-13-4,5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8, 8-12- 3, 4-12-7,7-12-4, 5-12-6, 6-12-5, 4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1,1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2,1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4,4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8,8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2,3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4,5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5,6-11-7, or 7-11-6. The numbers indicate the number of nucleosides in X,Y, and Z regions in the 5′-X-Y-Z-3′ gapmer.

In some embodiments, one or more nucleosides in the 5′wing region of agapmer (X in the 5′-X-Y-Z-3′ formula) or the 3′wing region of a gapmer(Z in the 5′-X-Y-Z-3′ formula) are modified nucleotides (e.g.,high-affinity modified nucleosides). In some embodiments, the modifiednucleoside (e.g., high-affinity modified nucleosides) is a 2′-modifiednucleoside. In some embodiments, the 2′-modified nucleoside is a 2′-4′bicyclic nucleoside or a non-bicyclic 2′-modified nucleoside. In someembodiments, the high-affinity modified nucleoside is a 2′-4′ bicyclicnucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2′-modifiednucleoside (e.g., 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O-N-methylacetamido (2′-O-NMA)).

In some embodiments, one or more nucleosides in the 5′wing region of agapmer (X in the 5′-X-Y-Z-3′ formula) are high-affinity modifiednucleosides. In some embodiments, each nucleoside in the 5′wing regionof the gapmer (X in the 5′-X-Y-Z-3′ formula) is a high-affinity modifiednucleoside. In some embodiments, one or more nucleosides in the 3′wingregion of a gapmer (Z in the 5′-X-Y-Z-3′ formula) are high-affinitymodified nucleosides. In some embodiments, each nucleoside in the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) is a high-affinitymodified nucleoside. In some embodiments, one or more nucleosides in the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) arehigh-affinity modified nucleosides and one or more nucleosides in the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) arehigh-affinity modified nucleosides. In some embodiments, each nucleosidein the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) is ahigh-affinity modified nucleoside and each nucleoside in the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) is high-affinitymodified nucleoside.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X-Y-Z-3′formula) comprises the same high affinity nucleosides as the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula). For example, the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) may compriseone or more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or2′-O-Me). In another example, the 5′wing region of the gapmer (X in the5′-X-Y-Z-3′ formula) and the 3′wing region of the gapmer (Z in the5′-X-Y-Z-3′ formula) may comprise one or more 2′-4′ bicyclic nucleosides(e.g., LNA or cEt). In some embodiments, each nucleoside in the 5′wingregion of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) is a non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me). In some embodiments,each nucleoside in the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) and the 3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′formula) is a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ configuration,wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein each nucleoside in X and Z is anon-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me) and eachnucleoside in Y is a 2′-deoxyribonucleoside. In some embodiments, thegapmer comprises a 5′-X-Y-Z-3′ configuration, wherein X and Z isindependently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in lengthand Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, whereineach nucleoside in X and Z is a 2′-4′ bicyclic nucleosides (e.g., LNA orcEt) and each nucleoside in Y is a 2′-deoxyribonucleoside. In someembodiments, the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) comprises different high affinity nucleosides as the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula). For example, the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) may compriseone or more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or2′-O-Me) and the 3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′formula) may comprise one or more 2′-4′ bicyclic nucleosides (e.g., LNAor cEt). In another example, the 3′wing region of the gapmer (Z in the5′-X-Y-Z-3′ formula) may comprise one or more non-bicyclic 2′-modifiednucleosides (e.g., 2′-MOE or 2′-O-Me) and the 5′wing region of thegapmer (X in the 5′-X-Y-Z-3′ formula) may comprise one or more 2′-4′bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ configuration,wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein each nucleoside in X is a non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me), each nucleoside in Zis a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), and each nucleosidein Y is a 2′-deoxyribonucleoside. In some embodiments, the gapmercomprises a 5′-X-Y-Z-3′ configuration, wherein X and Z is independently1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10(e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleosidein X is a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), each nucleosidein Z is a non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me)and each nucleoside in Y is a 2′-deoxyribonucleoside.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X-Y-Z-3′formula) comprises one or more non-bicyclic 2′-modified nucleosides(e.g., 2′-MOE or 2′-O-Me) and one or more 2′-4′ bicyclic nucleosides(e.g., LNA or cEt). In some embodiments, the 3′wing region of the gapmer(Z in the 5′-X-Y-Z-3′ formula) comprises one or more non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me) and one or more 2′-4′bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) comprise oneor more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me)and one or more 2′-4′ bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ configuration,wherein X and Z is independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3,4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X (the 5′ mostposition is position 1) is a non-bicyclic 2′-modified nucleoside (e.g.,2′-MOE or 2′-O-Me), wherein the rest of the nucleosides in both X and Zare 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), and wherein eachnucleoside in Y is a 2′deoxyribonucleoside. In some embodiments, thegapmer comprises a 5′-X-Y-Z-3′ configuration, wherein X and Z isindependently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length andY is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein atleast one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3,4, 5, 6, or 7 in Z (the 5′ most position is position 1) is anon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me), whereinthe rest of the nucleosides in both X and Z are 2′-4′ bicyclicnucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a2′deoxyribonucleoside. In some embodiments, the gapmer comprises a5′-X-Y-Z-3′ configuration, wherein X and Z is independently 2-7 (e.g.,2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8,9, or 10) nucleosides in length, wherein at least one but not all (e.g.,1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X and atleast one of positions but not all (e.g., 1, 2, 3, 4, 5, or 6) 1, 2, 3,4, 5, 6, or 7 in Z (the 5′ most position is position 1) is anon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me), whereinthe rest of the nucleosides in both X and Z are 2′-4′ bicyclicnucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a2′deoxyribonucleoside.

Non-limiting examples of gapmers configurations with a mix ofnon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me) and 2′-4′bicyclic nucleosides (e.g., LNA or cEt) in the 5′wing region of thegapmer (X in the 5′-X-Y-Z-3′ formula) and/or the 3′wing region of thegapmer (Z in the 5′-X-Y-Z-3′ formula) include: BBB-(D)n-BBBAA;KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE;LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA;BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BABA-(D)n-ABAB;KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK;LELE-(D)n-ELEL; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL;BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; ABAB-(D)n-ABAB;AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK;ELEL-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL;EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; AABB-(D)n-BBAA;BBAA-(D)n-AABB; AAKK-(D)n-KKAA; AALL-(D)n-LLAA; EEBB-(D)n-BBEE;EEKK-(D)n-KKEE; EELL-(D)n-LLEE; AABB-(D)n-BBAA; AAKK-(D)n-KKAA;AALL-(D)n-LLAA; EEBB-(D)n-BBEE; EEKK-(D)n-KKEE; EELL-(D)n-LLEE;BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA; BBB-(D)n-BBE; KKK-(D)n-KKE;LLL-(D)n-LLE; BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA; BBB-(D)n-BBE;KKK-(D)n-KKE; LLL-(D)n-LLE; BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA;BBB-(D)n-BBE; KKK-(D)n-KKE; LLL-(D)n-LLE; ABBB-(D)n-BBBA;AKKK-(D)n-KKKA; ALLL-(D)n-LLLA; EBBB-(D)n-BBBE; EKKK-(D)n-KKKE;ELLL-(D)n-LLLE; ABBB-(D)n-BBBA; AKKK-(D)n-KKKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE; EKKK-(D)n-KKKE; ELLL-(D)n-LLLE; ABBB-(D)n-BBBAA;AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA; EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE;ELLL-(D)n-LLLEE; ABBB-(D)n-BBBAA; AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE; ELLL-(D)n-LLLEE; AABBB-(D)n-BBB;AAKKK-(D)n-KKK; AALLL-(D)n-LLL; EEBBB-(D)n-BBB; EEKKK-(D)n-KKK;EELLL-(D)n-LLL; AABBB-(D)n-BBB; AAKKK-(D)n-KKK; AALLL-(D)n-LLL;EEBBB-(D)n-BBB; EEKKK-(D)n-KKK; EELLL-(D)n-LLL; AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA; AALLL-(D)n-LLLA; EEBBB-(D)n-BBBE; EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE; AABBB-(D)n-BBBA; AAKKK-(D)n-KKKA; AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE; EEKKK-(D)n-KKKE; EELLL-(D)n-LLLE; ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK; ALLAALLL-(D)n-LL; EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL; ABBAABB-(D)n-BB; AKKAAKK-(D)n-KK; ALLAALL-(D)n-LL;EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK; ELLEELL-(D)n-LL; ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK; ALLALLL-(D)n-LLL; EBBEBB-(D)n-BBB; EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL; ABBABB-(D)n-BBB; AKKAKK-(D)n-KKK; ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB; EKKEKK-(D)n-KKK; ELLELL-(D)n-LLL; EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEE; EK-(D)n-EEEEEEEEEE; EK-(D)n-EEEKK; K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE; K-(D)n-EEKEK; EK-(D)n-EEEEKEKE; EK-(D)n-EEEKEK;EEK-(D)n-KEEKE; EK-(D)n-EEKEK; EK-(D)n-KEEK; EEK-(D)n-EEEKEK;EK-(D)n-KEEEKEE; EK-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK.“A” nucleosides comprise a 2′-modified nucleoside; “B” represents a2′-4′ bicyclic nucleoside; “K” represents a constrained ethyl nucleoside(cEt); “L” represents an LNA nucleoside; and “E” represents a 2′-MOEmodified ribonucleoside; “D” represents a 2′-deoxyribonucleoside; “n”represents the length of the gap segment (Y in the 5′-X-Y-Z-3′configuration) and is an integer between 1-20.

In some embodiments, any one of the gapmers described herein comprisesone or more modified nucleoside linkages (e.g., a phosphorothioatelinkage) in each of the X, Y, and Z regions. In some embodiments, eachinternucleoside linkage in the any one of the gapmers described hereinis a phosphorothioate linkage. In some embodiments, each of the X, Y,and Z regions independently comprises a mix of phosphorothioate linkagesand phosphodiester linkages. In some embodiments, each internucleosidelinkage in the gap region Y is a phosphorothioate linkage, the 5′wingregion X comprises a mix of phosphorothioate linkages and phosphodiesterlinkages, and the 3′wing region Z comprises a mix of phosphorothioatelinkages and phosphodiester linkages.

i. Mixmers

In some embodiments, an oligonucleotide described herein may be a mixmeror comprise a mixmer sequence pattern. In general, mixmers areoligonucleotides that comprise both naturally and non-naturallyoccurring nucleosides or comprise two different types of non-naturallyoccurring nucleosides typically in an alternating pattern. Mixmersgenerally have higher binding affinity than unmodified oligonucleotidesand may be used to specifically bind a target molecule, e.g., to block abinding site on the target molecule. Generally, mixmers do not recruitan RNase to the target molecule and thus do not promote cleavage of thetarget molecule. Such oligonucleotides that are incapable of recruitingRNase H have been described, for example, see WO2007/112754 orWO2007/112753.

In some embodiments, the mixmer comprises or consists of a repeatingpattern of nucleoside analogues and naturally occurring nucleosides, orone type of nucleoside analogue and a second type of nucleosideanalogue. However, a mixmer need not comprise a repeating pattern andmay instead comprise any arrangement of modified nucleoside s andnaturally occurring nucleoside s or any arrangement of one type ofmodified nucleoside and a second type of modified nucleoside. Therepeating pattern, may, for instance be every second or every thirdnucleoside is a modified nucleoside, such as LNA, and the remainingnucleosides are naturally occurring nucleosides, such as DNA, or are a2′ substituted nucleoside analogue such as 2′-MOE or 2′ fluoroanalogues, or any other modified nucleoside described herein. It isrecognized that the repeating pattern of modified nucleoside, such asLNA units, may be combined with modified nucleoside at fixedpositions—e.g. at the 5′ or 3′ termini.

In some embodiments, a mixmer does not comprise a region of more than 5,more than 4, more than 3, or more than 2 consecutive naturally occurringnucleosides, such as DNA nucleosides. In some embodiments, the mixmercomprises at least a region consisting of at least two consecutivemodified nucleoside, such as at least two consecutive LNAs. In someembodiments, the mixmer comprises at least a region consisting of atleast three consecutive modified nucleoside units, such as at leastthree consecutive LNAs.

In some embodiments, the mixmer does not comprise a region of more than7, more than 6, more than 5, more than 4, more than 3, or more than 2consecutive nucleoside analogues, such as LNAs. In some embodiments, LNAunits may be replaced with other nucleoside analogues, such as thosereferred to herein.

Mixmers may be designed to comprise a mixture of affinity enhancingmodified nucleosides, such as in non-limiting example LNA nucleosidesand 2′-O-Me nucleosides. In some embodiments, a mixmer comprisesmodified internucleoside linkages (e.g., phosphorothioateinternucleoside linkages or other linkages) between at least two, atleast three, at least four, at least five or more nucleosides.

A mixmer may be produced using any suitable method. Representative U.S.patents, U.S. patent publications, and PCT publications that teach thepreparation of mixmers include U.S. patent publication Nos.US20060128646, US20090209748, US20090298916, US20110077288, andUS20120322851, and U.S. Pat. No. 7,687,617.

In some embodiments, a mixmer comprises one or more morpholinonucleosides. For example, in some embodiments, a mixmer may comprisemorpholino nucleosides mixed (e.g., in an alternating manner) with oneor more other nucleosides (e.g., DNA, RNA nucleosides) or modifiednucleosides (e.g., LNA, 2′-O-Me nucleosides).

In some embodiments, mixmers are useful for splice correcting or exonskipping, for example, as reported in Touznik A., et al., LNA/DNAmixmer-based antisense oligonucleotides correct alternative splicing ofthe SMN2 gene and restore SMN protein expression in type 1 SMAfibroblasts Scientific Reports, volume 7, Article number: 3672 (2017),Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA)-UridinePhosphoramidite, and Exon Skipping Using MNA/2′-O-Methyl MixmerAntisense Oligonucleotide, Molecules 2016, 21, 1582, the contents ofeach which are incorporated herein by reference.

j. RNA Interference (RNAi)

In some embodiments, oligonucleotides provided herein may be in the formof small interfering RNAs (siRNA), also known as short interfering RNAor silencing RNA. SiRNA, is a class of double-stranded RNA molecules,typically about 20-25 base pairs in length that target nucleic acids(e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway incells. Specificity of siRNA molecules may be determined by the bindingof the antisense strand of the molecule to its target RNA. EffectivesiRNA molecules are generally less than 30 to 35 base pairs in length toprevent the triggering of non-specific RNA interference pathways in thecell via the interferon response, although longer siRNA can also beeffective. In some embodiments, the siRNA molecules are 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or more base pairs in length. In some embodiments,the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 basepairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs inlength, 19 to 21 base pairs in length, 21 to 23 base pairs in length.

Following selection of an appropriate target RNA sequence, siRNAmolecules that comprise a nucleotide sequence complementary to all or aportion of the target sequence, i.e. an antisense sequence, can bedesigned and prepared using appropriate methods (see, e.g., PCTPublication Number WO 2004/016735; and U.S. Patent Publication Nos.2004/0077574 and 2008/0081791). The siRNA molecule can be doublestranded (i.e. a dsRNA molecule comprising an antisense strand and acomplementary sense strand that hybridizes to form the dsRNA) orsingle-stranded (i.e. a ssRNA molecule comprising just an antisensestrand). The siRNA molecules can comprise a duplex, asymmetric duplex,hairpin or asymmetric hairpin secondary structure, havingself-complementary sense and antisense strands.

In some embodiments, the antisense strand of the siRNA molecule is 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In someembodiments, the antisense strand is 8 to 50 nucleotides in length, 8 to40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 19 to 21 nucleotides in length, 21 to 23nucleotides in lengths.

In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In someembodiments, the sense strand is 8 to 50 nucleotides in length, 8 to 40nucleotides in length, 8 to 30 nucleotides in length, 10 to 15nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 19 to 21 nucleotides in length, 21 to 23nucleotides in lengths.

In some embodiments, siRNA molecules comprise an antisense strandcomprising a region of complementarity to a target region in a targetmRNA. In some embodiments, the region of complementarity is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% complementary to a target region in a targetmRNA. In some embodiments, the target region is a region of consecutivenucleotides in the target mRNA. In some embodiments, a complementarynucleotide sequence need not be 100% complementary to that of its targetto be specifically hybridizable or specific for a target RNA sequence.

In some embodiments, siRNA molecules comprise an antisense strand thatcomprises a region of complementarity to a target RNA sequence and theregion of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40,or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In someembodiments, a region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 nucleotides in length. In some embodiments, the region ofcomplementarity is complementary with at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25 or more consecutive nucleotides of a target RNA sequence. Insome embodiments, siRNA molecules comprise a nucleotide sequence thatcontains no more than 1, 2, 3, 4, or 5 base mismatches compared to theportion of the consecutive nucleotides of target RNA sequence. In someembodiments, siRNA molecules comprise a nucleotide sequence that has upto 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

In some embodiments, siRNA molecules comprise an antisense strandcomprising a nucleotide sequence that is complementary (e.g., at least85%, at least 90%, at least 95%, or 100%) to the target RNA sequence ofthe oligonucleotides provided herein. In some embodiments, siRNAmolecules comprise an antisense strand comprising a nucleotide sequencethat is at least 85%, at least 90%, at least 95%, or 100% identical tothe oligonucleotides provided herein. In some embodiments, siRNAmolecules comprise an antisense strand comprising at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25 or more consecutive nucleotides of theoligonucleotides provided herein.

Double-stranded siRNA may comprise sense and anti-sense RNA strands thatare the same length or different lengths. Double-stranded siRNAmolecules can also be assembled from a single oligonucleotide in astem-loop structure, wherein self-complementary sense and antisenseregions of the siRNA molecule are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s), as well as circularsingle-stranded RNA having two or more loop structures and a stemcomprising self-complementary sense and antisense strands, wherein thecircular RNA can be processed either in vivo or in vitro to generate anactive siRNA molecule capable of mediating RNAi. Small hairpin RNA(shRNA) molecules thus are also contemplated herein. These moleculescomprise a specific antisense sequence in addition to the reversecomplement (sense) sequence, typically separated by a spacer or loopsequence. Cleavage of the spacer or loop provides a single-stranded RNAmolecule and its reverse complement, such that they may anneal to form adsRNA molecule (optionally with additional processing steps that mayresult in addition or removal of one, two, three or more nucleotidesfrom the 3′ end and/or (e.g., and) the 5′ end of either or bothstrands). A spacer can be of a sufficient length to permit the antisenseand sense sequences to anneal and form a double-stranded structure (orstem) prior to cleavage of the spacer (and, optionally, subsequentprocessing steps that may result in addition or removal of one, two,three, four, or more nucleotides from the 3′ end and/or (e.g., and) the5′ end of either or both strands). A spacer sequence is may be anunrelated nucleotide sequence that is situated between two complementarynucleotide sequence regions which, when annealed into a double-strandednucleic acid, comprise a shRNA.

The overall length of the siRNA molecules can vary from about 14 toabout 100 nucleotides depending on the type of siRNA molecule beingdesigned. Generally between about 14 and about 50 of these nucleotidesare complementary to the RNA target sequence, i.e. constitute thespecific antisense sequence of the siRNA molecule. For example, when thesiRNA is a double- or single-stranded siRNA, the length can vary fromabout 14 to about 50 nucleotides, whereas when the siRNA is a shRNA orcircular molecule, the length can vary from about 40 nucleotides toabout 100 nucleotides.

An siRNA molecule may comprise a 3′ overhang at one end of the molecule,The other end may be blunt-ended or have also an overhang (5′ or 3′).When the siRNA molecule comprises an overhang at both ends of themolecule, the length of the overhangs may be the same or different. Inone embodiment, the siRNA molecule of the present disclosure comprises3′ overhangs of about 1 to about 3 nucleotides on both ends of themolecule. In some embodiments, the siRNA molecule comprises 3′ overhangsof about 1 to about 3 nucleotides on the sense strand. In someembodiments, the siRNA molecule comprises 3′ overhangs of about 1 toabout 3 nucleotides on the antisense strand. In some embodiments, thesiRNA molecule comprises 3′ overhangs of about 1 to about 3 nucleotideson both the sense strand and the antisense strand.

In some embodiments, the siRNA molecule comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the siRNA molecule comprises one or more modifiednucleotides and/or (e.g., and) one or more modified internucleotidelinkages. In some embodiments, the modified nucleotide is a modifiedsugar moiety (e.g. a 2′ modified nucleotide). In some embodiments, thesiRNA molecule comprises one or more 2′ modified nucleotides, e.g., a2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl(2′-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido(2′-O-NMA). In some embodiments, each nucleotide of the siRNA moleculeis a modified nucleotide (e.g., a 2′-modified nucleotide). In someembodiments, the siRNA molecule comprises one or more phosphorodiamidatemorpholinos. In some embodiments, each nucleotide of the siRNA moleculeis a phosphorodiamidate morpholino.

In some embodiments, the siRNA molecule contains a phosphorothioate orother modified internucleotide linkage. In some embodiments, the siRNAmolecule comprises phosphorothioate internucleoside linkages. In someembodiments, the siRNA molecule comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the siRNA molecule comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, the siRNA molecule comprises modified internucleotidelinkages at the first, second, and/or (e.g., and) third internucleosidelinkage at the 5′ or 3′ end of the siRNA molecule.

In some embodiments, the modified internucleotide linkages arephosphorus-containing linkages. In some embodiments,phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Any of the modified chemistries or formats of siRNA molecules describedherein can be combined with each other. For example, one, two, three,four, five, or more different types of modifications can be includedwithin the same siRNA molecule.

In some embodiments, the antisense strand comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the antisense strand comprises one or more modifiednucleotides and/or (e.g., and) one or more modified internucleotidelinkages. In some embodiments, the modified nucleotide comprises amodified sugar moiety (e.g. a 2′ modified nucleotide). In someembodiments, the antisense strand comprises one or more 2′ modifiednucleotides, e.g., a 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O-N-methylacetamido (2′-O-NMA). In some embodiments, each nucleotideof the antisense strand is a modified nucleotide (e.g., a 2′-modifiednucleotide). In some embodiments, the antisense strand comprises one ormore phosphorodiamidate morpholinos. In some embodiments, the antisensestrand is a phosphorodiamidate morpholino oligomer (PMO).

In some embodiments, antisense strand contains a phosphorothioate orother modified internucleotide linkage. In some embodiments, theantisense strand comprises phosphorothioate internucleoside linkages. Insome embodiments, the antisense strand comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the antisense strand comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, the antisense strand comprises modified internucleotidelinkages at the first, second, and/or (e.g., and) third internucleosidelinkage at the 5′ or 3′ end of the siRNA molecule. In some embodiments,the modified internucleotide linkages are phosphorus-containinglinkages. In some embodiments, phosphorus-containing linkages that maybe used include, but are not limited to, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatescomprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050.

Any of the modified chemistries or formats of the antisense stranddescribed herein can be combined with each other. For example, one, two,three, four, five, or more different types of modifications can beincluded within the same antisense strand.

In some embodiments, the sense strand comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the sense strand comprises one or more modified nucleotidesand/or (e.g., and) one or more modified internucleotide linkages. Insome embodiments, the modified nucleotide is a modified sugar moiety(e.g. a 2′ modified nucleotide). In some embodiments, the sense strandcomprises one or more 2′ modified nucleotides, e.g., a 2′-deoxy,2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O-N-methylacetamido (2′-O-NMA). In someembodiments, each nucleotide of the sense strand is a modifiednucleotide (e.g., a 2′-modified nucleotide). In some embodiments, thesense strand comprises one or more phosphorodiamidate morpholinos. Insome embodiments, the antisense strand is a phosphorodiamidatemorpholino oligomer (PMO). In some embodiments, the sense strandcontains a phosphorothioate or other modified internucleotide linkage.In some embodiments, the sense strand comprises phosphorothioateinternucleoside linkages. In some embodiments, the sense strandcomprises phosphorothioate internucleoside linkages between at least twonucleotides. In some embodiments, the sense strand comprisesphosphorothioate internucleoside linkages between all nucleotides. Forexample, in some embodiments, the sense strand comprises modifiedinternucleotide linkages at the first, second, and/or (e.g., and) thirdinternucleoside linkage at the 5′ or 3′ end of the sense strand.

In some embodiments, the modified internucleotide linkages arephosphorus-containing linkages. In some embodiments,phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Any of the modified chemistries or formats of the sense strand describedherein can be combined with each other. For example, one, two, three,four, five, or more different types of modifications can be includedwithin the same sense strand.

In some embodiments, the antisense or sense strand of the siRNA moleculecomprises modifications that enhance or reduce RNA-induced silencingcomplex (RISC) loading. In some embodiments, the antisense strand of thesiRNA molecule comprises modifications that enhance RISC loading. Insome embodiments, the sense strand of the siRNA molecule comprisesmodifications that reduce RISC loading and reduce off-target effects. Insome embodiments, the antisense strand of the siRNA molecule comprises a2′-O-methoxyethyl (2′-MOE) modification. The addition of the2′-O-methoxyethyl (2′-MOE) group at the cleavage site improves both thespecificity and silencing activity of siRNAs by facilitating theoriented RNA-induced silencing complex (RISC) loading of the modifiedstrand, as described in Song et al., (2017) Mol Ther Nucleic Acids9:242-250, incorporated herein by reference in its entirety. In someembodiments, the antisense strand of the siRNA molecule comprises a2′-OMe-phosphorodithioate modification, which increases RISC loading asdescribed in Wu et al., (2014) Nat Commun 5:3459, incorporated herein byreference in its entirety.

In some embodiments, the sense strand of the siRNA molecule comprises a5′-morpholino, which reduces RISC loading of the sense strand andimproves antisense strand selection and RNAi activity, as described inKumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, incorporatedherein by reference in its entirety. In some embodiments, the sensestrand of the siRNA molecule is modified with a synthetic RNA-like highaffinity nucleotide analogue, Locked Nucleic Acid (LNA), which reducesRISC loading of the sense strand and further enhances antisense strandincorporation into RISC, as described in Elman et al., (2005) NucleicAcids Res. 33(1): 439-447, incorporated herein by reference in itsentirety. In some embodiments, the sense strand of the siRNA moleculecomprises a 5′ unlocked nucleic acid (UNA) modification, which reduceRISC loading of the sense strand and improve silencing potency of theantisense strand, as described in Snead et al., (2013) Mol Ther NucleicAcids 2(7):e103, incorporated herein by reference in its entirety. Insome embodiments, the sense strand of the siRNA molecule comprises a5-nitroindole modification, which decreased the RNAi potency of thesense strand and reduces off-target effects as described in Zhang etal., (2012) Chembiochem 13(13):1940-1945, incorporated herein byreference in its entirety. In some embodiments, the sense strandcomprises a 2′-O′methyl (2′-0-Me) modification, which reduces RISCloading and the off-target effects of the sense strand, as described inZheng et al., FASEB (2013) 27(10): 4017-4026, incorporated herein byreference in its entirety. In some embodiments, the sense strand of thesiRNA molecule is fully substituted with morpholino, 2′-MOE or 2′-O-Meresidues, and are not recognized by RISC as described in Kole et al.,(2012) Nature reviews. Drug Discovery 11(2):125-140, incorporated hereinby reference in its entirety. In some embodiments the antisense strandof the siRNA molecule comprises a 2′-MOE modification and the sensestrand comprises an 2′-O-Me modification (see e.g., Song et al., (2017)Mol Ther Nucleic Acids 9:242-250). In some embodiments at least one(e.g., at least 2, at least 3, at least 4, at least 5, at least 10)siRNA molecule is linked (e.g., covalently) to a muscle-targeting agent.In some embodiments, the muscle-targeting agent may comprise, or consistof, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), alipid (e.g., a microvesicle), or a sugar moiety (e.g., apolysaccharide). In some embodiments, the muscle-targeting agent is anantibody. In some embodiments, the muscle-targeting agent is ananti-transferrin receptor antibody (e.g., any one of the anti-TfRantibodies provided herein). In some embodiments, the muscle-targetingagent may be linked to the 5′ end of the sense strand of the siRNAmolecule. In some embodiments, the muscle-targeting agent may be linkedto the 3′ end of the sense strand of the siRNA molecule. In someembodiments, the muscle-targeting agent may be linked internally to thesense strand of the siRNA molecule. In some embodiments, themuscle-targeting agent may be linked to the 5′ end of the antisensestrand of the siRNA molecule. In some embodiments, the muscle-targetingagent may be linked to the 3′ end of the antisense strand of the siRNAmolecule. In some embodiments, the muscle-targeting agent may be linkedinternally to the antisense strand of the siRNA molecule.

k. microRNA (miRNAs)

In some embodiments, an oligonucleotide may be a microRNA (miRNA).MicroRNAs (referred to as “miRNAs”) are small non-coding RNAs, belongingto a class of regulatory molecules that control gene expression bybinding to complementary sites on a target RNA transcript. Typically,miRNAs are generated from large RNA precursors (termed pri-miRNAs) thatare processed in the nucleus into approximately 70 nucleotidepre-miRNAs, which fold into imperfect stem-loop structures. Thesepre-miRNAs typically undergo an additional processing step within thecytoplasm where mature miRNAs of 18-25 nucleotides in length are excisedfrom one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.

As used herein, miRNAs including pri-miRNA, pre-miRNA, mature miRNA orfragments of variants thereof that retain the biological activity ofmature miRNA. In one embodiment, the size range of the miRNA can be from21 nucleotides to 170 nucleotides. In one embodiment the size range ofthe miRNA is from 70 to 170 nucleotides in length. In anotherembodiment, mature miRNAs of from 21 to 25 nucleotides in length can beused.

l. Aptamers

In some embodiments, oligonucleotides provided herein may be in the formof aptamers. Generally, in the context of molecular payloads, aptamer isany nucleic acid that binds specifically to a target, such as a smallmolecule, protein, nucleic acid in a cell. In some embodiments, theaptamer is a DNA aptamer or an RNA aptamer. In some embodiments, anucleic acid aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA).It is to be understood that a single-stranded nucleic acid aptamer mayform helices and/or (e.g., and) loop structures. The nucleic acid thatforms the nucleic acid aptamer may comprise naturally occurringnucleotides, modified nucleotides, naturally occurring nucleotides withhydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., aPEG linker) inserted between one or more nucleotides, modifiednucleotides with hydrocarbon or PEG linkers inserted between one or morenucleotides, or a combination of thereof. Exemplary publications andpatents describing aptamers and method of producing aptamers include,e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos.5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249;5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCTapplication WO 99/31275, each incorporated herein by reference.

m. Ribozymes

In some embodiments, oligonucleotides provided herein may be in the formof a ribozyme. A ribozyme (ribonucleic acid enzyme) is a molecule,typically an RNA molecule, that is capable of performing specificbiochemical reactions, similar to the action of protein enzymes.Ribozymes are molecules with catalytic activities including the abilityto cleave at specific phosphodiester linkages in RNA molecules to whichthey have hybridized, such as mRNAs, RNA-containing substrates, lncRNAs,and ribozymes, themselves.

Ribozymes may assume one of several physical structures, one of which iscalled a “hammerhead.” A hammerhead ribozyme is composed of a catalyticcore containing nine conserved bases, a double-stranded stem and loopstructure (stem-loop II), and two regions complementary to the targetRNA flanking regions the catalytic core. The flanking regions enable theribozyme to bind to the target RNA specifically by formingdouble-stranded stems I and III. Cleavage occurs in cis (i.e., cleavageof the same RNA molecule that contains the hammerhead motif) or in trans(cleavage of an RNA substrate other than that containing the ribozyme)next to a specific ribonucleotide triplet by a transesterificationreaction from a 3′, 5′-phosphate diester to a 2′, 3′-cyclic phosphatediester. Without wishing to be bound by theory, it is believed that thiscatalytic activity requires the presence of specific, highly conservedsequences in the catalytic region of the ribozyme.

Modifications in ribozyme structure have also included the substitutionor replacement of various non-core portions of the molecule withnon-nucleotidic molecules. For example, Benseler et al. (J. Am. Chem.Soc. (1993) 115:8483-8484) disclosed hammerhead-like molecules in whichtwo of the base pairs of stem II, and all four of the nucleotides ofloop II were replaced with non-nucleoside linkers based on hexaethyleneglycol, propanediol, bis(triethylene glycol) phosphate,tris(propanediol)bisphosphate, or bis(propanediol) phosphate. Ma et al.(Biochem. (1993) 32:1751-1758; Nucleic Acids Res. (1993) 21:2585-2589)replaced the six nucleotide loop of the TAR ribozyme hairpin withnon-nucleotidic, ethylene glycol-related linkers. Thomson et al.(Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear,non-nucleotidic linkers of 13, 17, and 19 atoms in length.

Ribozyme oligonucleotides can be prepared using well known methods (see,e.g., PCT Publications WO9118624; WO9413688; WO9201806; and WO 92/07065;and U.S. Pat. Nos. 5,436,143 and 5,650,502) or can be purchased fromcommercial sources (e.g., US Biochemicals) and, if desired, canincorporate nucleotide analogs to increase the resistance of theoligonucleotide to degradation by nucleases in a cell. The ribozyme maybe synthesized in any known manner, e.g., by use of a commerciallyavailable synthesizer produced, e.g., by Applied Biosystems, Inc. orMilligen. The ribozyme may also be produced in recombinant vectors byconventional means. See, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (Current edition). The ribozyme RNA sequencesmaybe synthesized conventionally, for example, by using RNA polymerasessuch as T7 or SP6.

n. Guide Nucleic Acids

In some embodiments, oligonucleotides are guide nucleic acid, e.g.,guide RNA (gRNA) molecules. Generally, a guide RNA is a short syntheticRNA composed of (1) a scaffold sequence that binds to a nucleic acidprogrammable DNA binding protein (napDNAbp), such as Cas9, and (2) anucleotide spacer portion that defines the DNA target sequence (e.g.,genomic DNA target) to which the gRNA binds in order to bring thenucleic acid programmable DNA binding protein in proximity to the DNAtarget sequence. In some embodiments, the napDNAbp is a nucleicacid-programmable protein that forms a complex with (e.g., binds orassociates with) one or more RNA(s) that targets the nucleicacid-programmable protein to a target DNA sequence (e.g., a targetgenomic DNA sequence). In some embodiments, a nucleic acid-programmablenuclease, when in a complex with an RNA, may be referred to as anuclease:RNA complex. Guide RNAs can exist as a complex of two or moreRNAs, or as a single RNA molecule.

Guide RNAs (gRNAs) that exist as a single RNA molecule may be referredto as single-guide RNAs (sgRNAs), though gRNA is also used to refer toguide RNAs that exist as either single molecules or as a complex of twoor more molecules. Typically, gRNAs that exist as a single RNA speciescomprise two domains: (1) a domain that shares homology to a targetnucleic acid (i.e., directs binding of a Cas9 complex to the target);and (2) a domain that binds a Cas9 protein. In some embodiments, domain(2) corresponds to a sequence known as a tracrRNA and comprises astem-loop structure. In some embodiments, domain (2) is identical orhomologous to a tracrRNA as provided in Jinek et al., Science337:816-821 (2012), the entire contents of which is incorporated hereinby reference.

In some embodiments, a gRNA comprises two or more of domains (1) and(2), and may be referred to as an extended gRNA. For example, anextended gRNA will bind two or more Cas9 proteins and bind a targetnucleic acid at two or more distinct regions, as described herein. ThegRNA comprises a nucleotide sequence that complements a target site,which mediates binding of the nuclease/RNA complex to said target site,providing the sequence specificity of the nuclease:RNA complex. In someembodiments, the RNA-programmable nuclease is the (CRISPR-associatedsystem) Cas9 endonuclease, for example, Cas9 (Csn1) from Streptococcuspyogenes (see, e.g., “Complete genome sequence of an M1 strain ofStreptococcus pyogenes.” Ferretti J. J., McShan W. M., Ajdic D. J.,Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N.,Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., RenQ., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A.,McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNaseIII.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y.,Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonucleasein adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I.,Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), theentire contents of each of which are incorporated herein by reference.

o. Multimers

In some embodiments, molecular payloads may comprise multimers (e.g.,concatemers) of 2 or more oligonucleotides connected by a linker. Inthis way, in some embodiments, the oligonucleotide loading of a complexcan be increased beyond the available linking sites on a targeting agent(e.g., available thiol sites on an antibody) or otherwise tuned toachieve a particular payload loading content. Oligonucleotides in amultimer can be the same or different (e.g., targeting different genesor different sites on the same gene or products thereof).

In some embodiments, multimers comprise 2 or more oligonucleotideslinked together by a cleavable linker. However, in some embodiments,multimers comprise 2 or more oligonucleotides linked together by anon-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4,5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In someembodiments, a multimer comprises 2 to 5, 2 to 10 or 4 to 20oligonucleotides linked together.

In some embodiments, a multimer comprises 2 or more oligonucleotideslinked end-to-end (in a linear arrangement). In some embodiments, amultimer comprises 2 or more oligonucleotides linked end-to-end via aoligonucleotide based linker (e.g., poly-dT linker, an abasic linker).In some embodiments, a multimer comprises a 5′ end of oneoligonucleotide linked to a 3′ end of another oligonucleotide. In someembodiments, a multimer comprises a 3′ end of one oligonucleotide linkedto a 3′ end of another oligonucleotide. In some embodiments, a multimercomprises a 5′ end of one oligonucleotide linked to a 5′ end of anotheroligonucleotide. Still, in some embodiments, multimers can comprise abranched structure comprising multiple oligonucleotides linked togetherby a branching linker.

Further examples of multimers that may be used in the complexes providedherein are disclosed, for example, in US Patent Application Number2015/0315588 A1, entitled Methods of delivering multiple targetingoligonucleotides to a cell using cleavable linkers, which was publishedon Nov. 5, 2015; US Patent Application Number 2015/0247141 A1, entitledMultimeric Oligonucleotide Compounds, which was published on Sep. 3,2015, US Patent Application Number US 2011/0158937 A1, entitledImmunostimulatory Oligonucleotide Multimers, which was published on Jun.30, 2011; and U.S. Pat. No. 5,693,773, entitled Triplex-FormingAntisense Oligonucleotides Having Abasic Linkers Targeting Nucleic AcidsComprising Mixed Sequences Of Purines And Pyrimidines, which issued onDec. 2, 1997, the contents of each of which are incorporated herein byreference in their entireties.

ii. Small Molecules:

Any suitable small molecule may be used as a molecular payload, asdescribed herein. In some embodiments, the small molecule enhances exonskipping of DMD mutant sequences. In some embodiments, the smallmolecule is as described in US Patent Application PublicationUS20140080896A1, published Mar. 20, 2014, entitled “IDENTIFICATION OFSMALL MOLECULES THAT FACILITATE THERAPEUTIC EXON SKIPPING”. Furtherexamples of small molecule payloads are provided in U.S. Pat. No.9,982,260, issued May 29, 2018, entitled “Identification of structurallysimilar small molecules that enhance therapeutic exon skipping”. Forexample, in some embodiments, the small molecule is an enhancer of exonskipping such as perphenazine, flupentixol, zuclopenthixol orcorynanthine. In some embodiments, a small molecule enhancer of exonskipping inhibits the ryanodine receptor or calmodulin. In someembodiments, the small molecule is an H-Ras pathway inhibitor such asmanumycin A. In some embodiments, the small molecule is a suppressor ofstop codons and desensitizes ribosomes to premature stop codons. In someembodiments, the small molecule is ataluren, as described in McElroy S.P. et al. “A Lack of Premature Termination Codon Read Through Efficacyof PTC124 (Ataluren) in a Diverse Array of Reporter Assays.” PLOSBiology, published Jun. 25, 2013. In some embodiments, the smallmolecule is a corticosteroid, e.g., as described in Manzur, A. Y. et al.“Glucocorticoid corticosteroids for Duchenne muscular dystrophy”.Cochrane Database Syst Rev. 2004; (2):CD003725. In some embodiments, thesmall molecule upregulates the expression and/or (e.g., and) activity ofgenes that can replace the function of dystrophin, such as utrophin. Insome embodiments, a utrophin modulator is as described in InternationalPublication No. WO2007091106, published Aug. 16, 2007, entitled“TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY” and/or (e.g., and)International Publication No. WO/2017/168151, published Oct. 5, 2017,entitled “COMPOSITION FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY”.

iii. Peptides/Proteins

Any suitable peptide or protein may be used as a molecular payload, asdescribed herein. In some embodiments, a protein is an enzyme. In someembodiments, peptides or proteins may be produced, synthesized, and/or(e.g., and) derivatized using several methodologies, e.g. phagedisplayed peptide libraries, one-bead one-compound peptide libraries, orpositional scanning synthetic peptide combinatorial libraries. Exemplarymethodologies have been characterized in the art and are incorporated byreference (Gray, B. P. and Brown, K. C. “Combinatorial PeptideLibraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2,1020-1081.; Samoylova, T. I. and Smith, B. F. “Elucidation ofmuscle-binding peptides by phage display screening.” Muscle Nerve, 1999,22:4. 460-6.).

In some embodiments, a peptide may facilitate exon skipping in an mRNAexpressed from a mutated DMD allele. In some embodiments, a peptide maypromote the expression of functional dystrophin and/or (e.g., and) theexpression of a protein capable of functioning in place of dystrophin.In some embodiments, payload is a protein that is a functional fragmentof dystrophin, e.g. an amino acid segment of a functional dystrophinprotein.

In some embodiments, the peptide or protein comprises at least one zincfinger.

In some embodiments, the peptide or protein may comprise about 2-25amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10amino acids, or about 2-5 amino acids. The peptide or protein maycomprise naturally-occurring amino acids, e.g. cysteine, alanine, ornon-naturally-occurring or modified amino acids. Non-naturally occurringamino acids include β-amino acids, homo-amino acids, prolinederivatives, 3-substituted alanine derivatives, linear core amino acids,N-methyl amino acids, and others known in the art. In some embodiments,the peptide may be linear; in other embodiments, the peptide may becyclic, e.g. bicyclic.

iv. Nucleic Acid Constructs

Any suitable gene expression construct may be used as a molecularpayload, as described herein. In some embodiments, a gene expressionconstruct may be a vector or a cDNA fragment. In some embodiments, agene expression construct may be messenger RNA (mRNA). In someembodiments, a mRNA used herein may be a modified mRNA, e.g., asdescribed in U.S. Pat. No. 8,710,200, issued on Apr. 24, 2014, entitled“Engineered nucleic acids encoding a modified erythropoietin and theirexpression”. In some embodiments, a mRNA may comprise a 5′ methyl cap.In some embodiments, a mRNA may comprise a polyA tail, optionally of upto 160 nucleotides in length. A gene expression construct may encode asequence of a dystrophin protein, a dystrophin fragment, amini-dystrophin, a utrophin protein, or any protein that shares a commonfunction with dystrophin. In some embodiments, the gene expressionconstruct may be expressed, e.g., overexpressed, within the nucleus of amuscle cell. In some embodiments, the gene expression constructs encodesa protein that comprises at least one zinc finger. In some embodiments,the gene expression construct encodes a protein that promotes theexpression of dystrophin or a protein that shares function withdystrophin, e.g., utrophin. In some embodiments, the gene expressionconstruct encodes a gene editing enzyme. In some embodiments, the geneexpression construct is as described in U.S. Patent ApplicationPublication US20170368198A1, published Dec. 28, 2017, entitled“Optimized mini-dystrophin genes and expression cassettes and theiruse”; Duan D. “Myodys, a full-length dystrophin plasmid vector forDuchenne and Becker muscular dystrophy gene therapy.” Curr Opin Mol Ther2008; 10:86-94; and expression cassettes disclosed in Tang, Y. et al.,“AAV-directed muscular dystrophy gene therapy” Expert Opin Biol Ther.2010 March; 10(3):395-408; the contents of each of which areincorporated herein by reference in their entireties.

C. Linkers

Complexes described herein generally comprise a linker that connects anyone of the anti-TfR antibodies described herein to a molecular payload.A linker comprises at least one covalent bond. In some embodiments, alinker may be a single bond, e.g., a disulfide bond or disulfide bridge,that connects an anti-TfR antibody to a molecular payload. However, insome embodiments, a linker may connect any one of the anti-TfRantibodies described herein to a molecular payload through multiplecovalent bonds. In some embodiments, a linker may be a cleavable linker.However, in some embodiments, a linker may be a non-cleavable linker. Alinker is generally stable in vitro and in vivo, and may be stable incertain cellular environments. Additionally, generally a linker does notnegatively impact the functional properties of either the anti-TfRantibody or the molecular payload. Examples and methods of synthesis oflinkers are known in the art (see, e.g. Kline, T. et al. “Methods toMake Homogenous Antibody Drug Conjugates.” Pharmaceutical Research,2015, 32:11, 3480-3493.; Jain, N. et al. “Current ADC Linker Chemistry”Pharm Res. 2015, 32:11, 3526-3540.; McCombs, J. R. and Owen, S. C.“Antibody Drug Conjugates: Design and Selection of Linker, Payload andConjugation Chemistry” AAPS J. 2015, 17:2, 339-351.).

A precursor to a linker typically will contain two different reactivespecies that allow for attachment to both the anti-TfR antibody and amolecular payload. In some embodiments, the two different reactivespecies may be a nucleophile and/or (e.g., and) an electrophile. In someembodiments, a linker is connected to an anti-TfR antibody viaconjugation to a lysine residue or a cysteine residue of the anti-TfRantibody. In some embodiments, a linker is connected to a cysteineresidue of an anti-TfR antibody via a maleimide-containing linker,wherein optionally the maleimide-containing linker comprises amaleimidocaproyl or maleimidomethyl cyclohexane-1-carboxylate group. Insome embodiments, a linker is connected to a cysteine residue of ananti-TfR antibody or thiol functionalized molecular payload via a3-arylpropionitrile functional group. In some embodiments, a linker isconnected to a lysine residue of an anti-TfR antibody. In someembodiments, a linker is connected to an anti-TfR antibody and/or (e.g.,and) a molecular payload via an amide bond, a carbamate bond, ahydrazide, a triazole, a thioether, or a disulfide bond.

i. Cleavable Linkers

A cleavable linker may be a protease-sensitive linker, a pH-sensitivelinker, or a glutathione-sensitive linker. These linkers are generallycleavable only intracellularly and are preferably stable inextracellular environments, e.g. extracellular to a muscle cell.

Protease-sensitive linkers are cleavable by protease enzymatic activity.These linkers typically comprise peptide sequences and may be 2-10 aminoacids, about 2-5 amino acids, about 5-10 amino acids, about 10 aminoacids, about 5 amino acids, about 3 amino acids, or about 2 amino acidsin length. In some embodiments, a peptide sequence may comprisenaturally-occurring amino acids, e.g. cysteine, alanine, ornon-naturally-occurring or modified amino acids. Non-naturally occurringamino acids include β-amino acids, homo-amino acids, prolinederivatives, 3-substituted alanine derivatives, linear core amino acids,N-methyl amino acids, and others known in the art. In some embodiments,a protease-sensitive linker comprises a valine-citrulline oralanine-citrulline dipeptide sequence. In some embodiments, aprotease-sensitive linker can be cleaved by a lysosomal protease, e.g.cathepsin B, and/or (e.g., and) an endosomal protease.

A pH-sensitive linker is a covalent linkage that readily degrades inhigh or low pH environments. In some embodiments, a pH-sensitive linkermay be cleaved at a pH in a range of 4 to 6. In some embodiments, apH-sensitive linker comprises a hydrazone or cyclic acetal. In someembodiments, a pH-sensitive linker is cleaved within an endosome or alysosome.

In some embodiments, a glutathione-sensitive linker comprises adisulfide moiety. In some embodiments, a glutathione-sensitive linker iscleaved by a disulfide exchange reaction with a glutathione speciesinside a cell. In some embodiments, the disulfide moiety furthercomprises at least one amino acid, e.g. a cysteine residue.

In some embodiments, the linker is a Val-cit linker (e.g., as describedin U.S. Pat. No. 6,214,345, incorporated herein by reference). In someembodiments, before conjugation, the val-cit linker has a structure of:

In some embodiments, after conjugation, the val-cit linker has astructure of:

In some embodiments, the Val-cit linker is attached to a reactivechemical moiety (e.g., SPAAC for click chemistry conjugation). In someembodiments, before click chemistry conjugation, the val-cit linkerattached to a reactive chemical moiety (e.g., SPAAC for click chemistryconjugation) has the structure of:

wherein n is any number from 0-10. In some embodiments, n is 3.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated(e.g., via a different chemical moiety) to a molecular payload (e.g., anoligonucleotide). In some embodiments, the val-cit linker attached to areactive chemical moiety (e.g., SPAAC for click chemistry conjugation)and conjugated to a molecular payload (e.g., an oligonucleotide) has thestructure of (before click chemistry conjugation):

wherein n is any number from 0-10. In some embodiments, n is 3.

In some embodiments, after conjugation to a molecular payload (e.g., anoligonucleotide), the val-cit linker has a structure of:

wherein n is any number from 0-10, and wherein m is any number from0-10. In some embodiments, n is 3 and m is 4.

ii. Non-Cleavable Linkers

In some embodiments, non-cleavable linkers may be used. Generally, anon-cleavable linker cannot be readily degraded in a cellular orphysiological environment. In some embodiments, a non-cleavable linkercomprises an optionally substituted alkyl group, wherein thesubstitutions may include halogens, hydroxyl groups, oxygen species, andother common substitutions. In some embodiments, a linker may comprisean optionally substituted alkyl, an optionally substituted alkylene, anoptionally substituted arylene, a heteroarylene, a peptide sequencecomprising at least one non-natural amino acid, a truncated glycan, asugar or sugars that cannot be enzymatically degraded, an azide, analkyne-azide, a peptide sequence comprising a LPXT sequence, athioether, a biotin, a biphenyl, repeating units of polyethylene glycolor equivalent compounds, acid esters, acid amides, sulfamides, and/or(e.g., and) an alkoxy-amine linker. In some embodiments,sortase-mediated ligation will be utilized to covalently link ananti-TfR antibody comprising a LPXT sequence to a molecular payloadcomprising a (G). sequence (see, e.g. Proft T. Sortase-mediated proteinligation: an emerging biotechnology tool for protein modification andimmobilization. Biotechnol Lett. 2010, 32(1):1-10.).

In some embodiments, a linker may comprise a substituted alkylene, anoptionally substituted alkenylene, an optionally substituted alkynylene,an optionally substituted cycloalkylene, an optionally substitutedcycloalkenylene, an optionally substituted arylene, an optionallysubstituted heteroarylene further comprising at least one heteroatomselected from N, O, and S; an optionally substituted heterocyclylenefurther comprising at least one heteroatom selected from N, O, and S; animino, an optionally substituted nitrogen species, an optionallysubstituted oxygen species O, an optionally substituted sulfur species,or a poly(alkylene oxide), e.g. polyethylene oxide or polypropyleneoxide.

iii. Linker Conjugation

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload via a phosphate, thioether, ether,carbon-carbon, carbamate, or amide bond. In some embodiments, a linkeris connected to an oligonucleotide through a phosphate orphosphorothioate group, e.g. a terminal phosphate of an oligonucleotidebackbone. In some embodiments, a linker is connected to an anti-TfRantibody, through a lysine or cysteine residue present on the anti-TfRantibody.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by a cycloaddition reaction betweenan azide and an alkyne to form a triazole, wherein the azide and thealkyne may be located on the anti-TfR antibody, molecular payload, orthe linker. In some embodiments, an alkyne may be a cyclic alkyne, e.g.,a cyclooctyne. In some embodiments, an alkyne may be bicyclononyne (alsoknown as bicyclo[6.1.0]nonyne or BCN) or substituted bicyclononyne. Insome embodiments, a cyclooctane is as described in International PatentApplication Publication WO2011136645, published on Nov. 3, 2011,entitled, “Fused Cyclooctyne Compounds And Their Use In Metal free ClickReactions”. In some embodiments, an azide may be a sugar or carbohydratemolecule that comprises an azide. In some embodiments, an azide may be6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In someembodiments, a sugar or carbohydrate molecule that comprises an azide isas described in International Patent Application PublicationWO2016170186, published on Oct. 27, 2016, entitled, “Process For TheModification Of A Glycoprotein Using A Glycosyltransferase That Is Or IsDerived From A β(1,4)-N-Acetylgalactosaminyltransferase”. In someembodiments, a cycloaddition reaction between an azide and an alkyne toform a triazole, wherein the azide and the alkyne may be located on theanti-TfR antibody, molecular payload, or the linker is as described inInternational Patent Application Publication WO2014065661, published onMay 1, 2014, entitled, “Modified antibody, antibody-conjugate andprocess for the preparation thereof”; or International PatentApplication Publication WO2016170186, published on Oct. 27, 2016,entitled, “Process For The Modification Of A Glycoprotein Using AGlycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”.

In some embodiments, a linker further comprises a spacer, e.g., apolyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g.,a HydraSpace™ spacer. In some embodiments, a spacer is as described inVerkade, J. M. M. et al., “A Polar Sulfamide Spacer SignificantlyEnhances the Manufacturability, Stability, and Therapeutic Index ofAntibody-Drug Conjugates”, Antibodies, 2018, 7, 12.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by the Diels-Alder reaction betweena dienophile and a diene/hetero-diene, wherein the dienophile and thediene/hetero-diene may be located on the anti-TfR antibody, molecularpayload, or the linker. In some embodiments a linker is connected to ananti-TfR antibody and/or (e.g., and) molecular payload by otherpericyclic reactions, e.g. ene reaction. In some embodiments, a linkeris connected to an anti-TfR antibody and/or (e.g., and) molecularpayload by an amide, thioamide, or sulfonamide bond reaction. In someembodiments, a linker is connected to an anti-TfR antibody and/or (e.g.,and) molecular payload by a condensation reaction to form an oxime,hydrazone, or semicarbazide group existing between the linker and theanti-TfR antibody and/or (e.g., and) molecular payload.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by a conjugate addition reactionsbetween a nucleophile, e.g. an amine or a hydroxyl group, carbonate, andan electrophile, e.g. a carboxylic acid or an aldehyde. In someembodiments, a nucleophile may exist on a linker and an electrophile mayexist on an anti-TfR antibody or molecular payload prior to a reactionbetween a linker and an anti-TfR antibody or molecular payload. In someembodiments, an electrophile may exist on a linker and a nucleophile mayexist on an anti-TfR antibody or molecular payload prior to a reactionbetween a linker and an anti-TfR antibody or molecular payload. In someembodiments, an electrophile may be an azide, pentafluorophenyl, asilicon centers, a carbonyl, a carboxylic acid, an anhydride, anisocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidylester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide,an episulfide, an aziridine, an aryl, an activated phosphorus center,and/or (e.g., and) an activated sulfur center. In some embodiments, anucleophile may be an optionally substituted alkene, an optionallysubstituted alkyne, an optionally substituted aryl, an optionallysubstituted heterocyclyl, a hydroxyl group, an amino group, analkylamino group, an anilido group, or a thiol group.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated tothe anti-TfR antibody by a structure of:

wherein m is any number from 0-10. In some embodiments, m is 4.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated to ananti-TfR antibody having a structure of:

wherein m is any number from 0-10. In some embodiments, m is 4.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) and conjugated toan anti-TfR antibody has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4.

In some embodiments, the val-cit linker used to covalently link ananti-TfR antibody and a molecular payload (e.g., an oligonucleotide) hasa structure of:

wherein n is 3 and m is 4.

In some embodiments, the val-cit linker that links the antibody and themolecular payload has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments,n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NHfrom an amine group of a lysine), S (e.g., S from a thiol group of acysteine), or O (e.g., O from a hydroxyl group of a serine, threonine,or tyrosine) of the antibody.

In some embodiments, the complex described herein has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4.

In structures formula (A), (B), (C), and (D), L1, in some embodiments,is a spacer that is a substituted or unsubstituted aliphatic,substituted or unsubstituted heteroaliphatic, substituted orunsubstituted carbocyclylene, substituted or unsubstitutedheterocyclylene, substituted or unsubstituted arylene, substituted orunsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—,—C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—,—NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—,—OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combinationthereof. In some embodiments, L1 is

wherein the piperazine moiety links to the oligonucleotide, wherein L2is

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide.

In some embodiments, L1 is

In some embodiments, L1 is linked to a 5′ phosphate of theoligonucleotide.

In some embodiments, L1 is optional (e.g., need not be present).

In some embodiments, any one of the complexes described herein has astructure of:

wherein n is 0-15 (e.g., 3) and m is 0-15 (e.g., 4).

C. Examples of Antibody-Molecular Payload Complexes

Further provided herein are non-limiting examples of complexescomprising any one the anti-TfR antibodies described herein covalentlylinked to any of the molecular payloads (e.g., an oligonucleotide)described herein. In some embodiments, the anti-TfR antibody (e.g., anyone of the anti-TfR antibodies provided in Table 2) is covalently linkedto a molecular payload (e.g., an oligonucleotide) via a linker. Any ofthe linkers described herein may be used. In some embodiments, if themolecular payload is an oligonucleotide, the linker is linked to the 5′end, the 3′ end, or internally of the oligonucleotide. In someembodiments, the linker is linked to the anti-TfR antibody via athiol-reactive linkage (e.g., via a cysteine in the anti-TfR antibody).In some embodiments, the linker (e.g., a Val-cit linker) is linked tothe antibody (e.g., an anti-TfR antibody described herein) via an aminegroup (e.g., via a lysine in the antibody). In some embodiments, themolecular payload is a DMD targeting oligonucleotide (e.g., anoligonucleotide listed in Table 14). In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046).

An example of a structure of a complex comprising an anti-TfR antibodycovalently linked to a molecular payload via a Val-cit linker isprovided below:

wherein the linker is linked to the antibody via a thiol-reactivelinkage (e.g., via a cysteine in the antibody). In some embodiments, themolecular payload is a DMD targeting oligonucleotide (e.g., anoligonucleotide listed in Table 14). In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046).

Another example of a structure of a complex comprising an anti-TfRantibody covalently linked to a molecular payload via a Val-cit linkeris provided below:

wherein n is a number between 0-10, wherein m is a number between 0-10,wherein the linker is linked to the antibody via an amine group (e.g.,on a lysine residue), and/or (e.g., and) wherein the linker is linked tothe oligonucleotide (e.g., at the 5′ end, 3′ end, or internally). Insome embodiments, the linker is linked to the antibody via a lysine, thelinker is linked to the oligonucleotide at the 5′ end, n is 3, and m is4. In some embodiments, the molecular payload is an oligonucleotidecomprising a sense strand and an antisense strand, and, the linker islinked to the sense strand or the antisense strand at the 5′ end or the3′ end.

It should be appreciated that antibodies can be linked to molecularpayloads with different stochiometries, a property that may be referredto as a drug to antibody ratios (DAR) with the “drug” being themolecular payload. In some embodiments, one molecular payload is linkedto an antibody (DAR=1). In some embodiments, two molecular payloads arelinked to an antibody (DAR=2). In some embodiments, three molecularpayloads are linked to an antibody (DAR=3). In some embodiments, fourmolecular payloads are linked to an antibody (DAR=4). In someembodiments, a mixture of different complexes, each having a differentDAR, is provided. In some embodiments, an average DAR of complexes insuch a mixture may be in a range of 1 to 3, 1 to 4, 1 to 5 or more. DARmay be increased by conjugating molecular payloads to different sites onan antibody and/or (e.g., and) by conjugating multimers to one or moresites on antibody. For example, a DAR of 2 may be achieved byconjugating a single molecular payload to two different sites on anantibody or by conjugating a dimer molecular payload to a single site ofan antibody.

In some embodiments, the complex described herein comprises an anti-TfRantibody described herein (e.g., the 3-A4, 3-M12, and 5-H12 antibodiesprovided in Table 2 in an IgG or Fab form) covalently linked to amolecular payload. In some embodiments, the complex described hereincomprises an anti-TfR antibody described herein (e.g., the 3-A4, 3-M12,and 5-H12 antibodies provided in Table 2 in an IgG or Fab form)covalently linked to molecular payload via a linker (e.g., a Val-citlinker). In some embodiments, the linker (e.g., a Val-cit linker) islinked to the antibody (e.g., an anti-TfR antibody described herein) viaa thiol-reactive linkage (e.g., via a cysteine in the antibody). In someembodiments, the linker (e.g., a Val-cit linker) is linked to theantibody (e.g., an anti-TfR antibody described herein) via an aminegroup (e.g., via a lysine in the antibody). In some embodiments, themolecular payload is a DMD targeting oligonucleotide (e.g., anoligonucleotide listed in Table 14). In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046).

In some embodiments, in any one of the examples of complexes describedherein, the molecular payload is a DMD targeting oligonucleotidecomprising a region of complementarity of at least 15 consecutivenucleotides to a target sequence provided by any one of SEQ ID NO:1242-2046. In some embodiments, in any one of the examples of complexesdescribed herein, the molecular payload is a DMD targetingoligonucleotide comprising a region of at least 15 consecutivenucleotides of any one of SEQ ID NO: 437-1241. In some embodiments, inany one of the examples of complexes described herein, the molecularpayload is a DMD targeting oligonucleotide comprising a region ofcomplementarity of at least 5 consecutive nucleotides of an ESE listedin Table 15. In some embodiments, in any one of the examples ofcomplexes described herein, the molecular payload is a DMD targetingoligonucleotide selected from the oligonucleotides listed in Table 14.In some embodiments, in any one of the examples of complexes describedherein, the molecular payload is a DMD targeting oligonucleotideselected from the oligonucleotides provided by any one of SEQ ID NO:437-1241, or complementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same asthe CDR-H1, CDR-H2, and CDR-H3 shown in Table 2; and a CDR-L1, a CDR-L2,and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shownin Table 2. In some embodiments, the molecular payload is a DMDtargeting oligonucleotide (e.g., an oligonucleotide listed in Table 14).In some embodiments, the molecular payload is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acidsequence of SEQ ID NO: 70. In some embodiments, the molecular payload isa DMD targeting oligonucleotide (e.g., an oligonucleotide listed inTable 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQID NO: 74. In some embodiments, the molecular payload is a DMD targetingoligonucleotide (e.g., an oligonucleotide listed in Table 14). In someembodiments, the molecular payload is an oligonucleotide provided by anyone of SEQ ID NO: 437-1241, or complementary to any one of SEQ ID NO:1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQID NO: 75. In some embodiments, the molecular payload is a DMD targetingoligonucleotide (e.g., an oligonucleotide listed in Table 14). In someembodiments, the molecular payload is an oligonucleotide provided by anyone of SEQ ID NO: 437-1241, or complementary to any one of SEQ ID NO:1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77, and a VL comprising the amino acid sequence of SEQ ID NO: 78. Insome embodiments, the molecular payload is a DMD targetingoligonucleotide (e.g., an oligonucleotide listed in Table 14). In someembodiments, the molecular payload is an oligonucleotide provided by anyone of SEQ ID NO: 437-1241, or complementary to any one of SEQ ID NO:1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQID NO: 80. In some embodiments, the molecular payload is a DMD targetingoligonucleotide (e.g., an oligonucleotide listed in Table 14). In someembodiments, the molecular payload is an oligonucleotide provided by anyone of SEQ ID NO: 437-1241, or complementary to any one of SEQ ID NO:1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85. In someembodiments, the molecular payload is a DMD targeting oligonucleotide(e.g., an oligonucleotide listed in Table 14). In some embodiments, themolecular payload is an oligonucleotide provided by any one of SEQ IDNO: 437-1241, or complementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the aminoacid sequence of SEQ ID NO: 89. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the aminoacid sequence of SEQ ID NO: 90. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 92 or SEQ ID NO: 94, and a light chain comprising the aminoacid sequence of SEQ ID NO: 95. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 92, and a light chain comprising the amino acid sequence ofSEQ ID NO: 93. In some embodiments, the molecular payload is a DMDtargeting oligonucleotide (e.g., an oligonucleotide listed in Table 14).In some embodiments, the molecular payload is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99 and a VL comprising theamino acid sequence of SEQ ID NO: 85. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 89. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 90. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 102 and a light chain comprising the amino acid sequence ofSEQ ID NO: 93. In some embodiments, the molecular payload is a DMDtargeting oligonucleotide (e.g., an oligonucleotide listed in Table 14).In some embodiments, the molecular payload is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 102 or SEQ ID NO: 103 and a light chain comprising the aminoacid sequence of SEQ ID NO: 95. In some embodiments, the molecularpayload is a DMD targeting oligonucleotide (e.g., an oligonucleotidelisted in Table 14). In some embodiments, the molecular payload is anoligonucleotide provided by any one of SEQ ID NO: 437-1241, orcomplementary to any one of SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 85; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 86 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 85; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 85; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 89; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 90; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 89; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 90; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 93; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 94 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 95; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 95; wherein thecomplex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 69 and a VL comprising the amino acid sequence ofin SEQ ID NO: 70; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence ofin SEO ID NO: 70: wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence ofin SEQ ID NO: 70; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence ofin SEQ ID NO: 74; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence ofin SEQ ID NO: 75; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence ofin SEQ ID NO: 74; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence ofin SEQ ID NO: 75; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence ofin SEQ ID NO: 78; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence ofin SEQ ID NO: 80; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence ofin SEQ ID NO: 80; wherein the complex has the structure of:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 97 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 85; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 98 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 85; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 99 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 85; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 100 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 89; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 100 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 90; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 89; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 90; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 102 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 93; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 103 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 95; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 102 and a light chain comprising the aminoacid sequence of in SEQ ID NO: 95; wherein the complex has the structureof:

wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is aDMD targeting oligonucleotide (e.g., an oligonucleotide listed in Table14). In some embodiments, the oligonucleotide is an oligonucleotideprovided by any one of SEQ ID NO: 437-1241, or complementary to any oneof SEQ ID NO: 1242-2046.

In some embodiments, in any one of the examples of complexes describedherein, L1 is any one of the spacers described herein.

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide, wherein L2is

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide.

In some embodiments, L1 is

In some embodiments, L1 is linked to a 5′ phosphate of theoligonucleotide.

In some embodiments, L1 is optional (e.g., need not be present).

III. Formulations

Complexes provided herein may be formulated in any suitable manner.Generally, complexes provided herein are formulated in a manner suitablefor pharmaceutical use. For example, complexes can be delivered to asubject using a formulation that minimizes degradation, facilitatesdelivery and/or (e.g., and) uptake, or provides another beneficialproperty to the complexes in the formulation. In some embodiments,provided herein are compositions comprising complexes andpharmaceutically acceptable carriers. Such compositions can be suitablyformulated such that when administered to a subject, either into theimmediate environment of a target cell or systemically, a sufficientamount of the complexes enter target muscle cells. In some embodiments,complexes are formulated in buffer solutions such as phosphate-bufferedsaline solutions, liposomes, micellar structures, and capsids.

It should be appreciated that, in some embodiments, compositions mayinclude separately one or more components of complexes provided herein(e.g., muscle-targeting agents, linkers, molecular payloads, orprecursor molecules of any one of them).

In some embodiments, complexes are formulated in water or in an aqueoussolution (e.g., water with pH adjustments). In some embodiments,complexes are formulated in basic buffered aqueous solutions (e.g.,PBS). In some embodiments, formulations as disclosed herein comprise anexcipient. In some embodiments, an excipient confers to a compositionimproved stability, improved absorption, improved solubility and/or(e.g., and) therapeutic enhancement of the active ingredient. In someembodiments, an excipient is a buffering agent (e.g., sodium citrate,sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g.,a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).

In some embodiments, a complex or component thereof (e.g.,oligonucleotide or antibody) is lyophilized for extending its shelf-lifeand then made into a solution before use (e.g., administration to asubject). Accordingly, an excipient in a composition comprising acomplex, or component thereof, described herein may be a lyoprotectant(e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone),or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, administration. Typically, the route of administration isintravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. In some embodiments, formulationsinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride in the composition. Sterileinjectable solutions can be prepared by incorporating the complexes in arequired amount in a selected solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization.

In some embodiments, a composition may contain at least about 0.1% ofthe a complex, or component thereof, or more, although the percentage ofthe active ingredient(s) may be between about 1% and about 80% or moreof the weight or volume of the total composition. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

IV. Methods of Use/Treatment

Complexes comprising a muscle-targeting agent covalently linked to amolecular payload as described herein are effective in treating asubject having a dystrophinopathy, e.g., Duchenne muscular dystrophy. Insome embodiments, complexes comprise a molecular payload that is anoligonucleotide, e.g., an antisense oligonucleotide that facilitatesexon skipping of an mRNA expressed from a mutated DMD allele.

In some embodiments, a subject may be a human subject, a non-humanprimate subject, a rodent subject, or any suitable mammalian subject. Insome embodiments, a subject may have Duchenne muscular dystrophy orother dystrophinopathy. In some embodiments, a subject has a mutated DMDallele, which may optionally comprise at least one mutation in a DMDexon that causes a frameshift mutation and leads to improper RNAsplicing/processing. In some embodiments, a subject is suffering fromsymptoms of a severe dystrophinopathy, e.g. muscle atrophy or muscleloss. In some embodiments, a subject has an asymptomatic increase inserum concentration of creatine phosphokinase (CK) and/or (e.g., and)muscle cramps with myoglobinuria. In some embodiments, a subject has aprogressive muscle disease, such as Duchenne or Becker musculardystrophy or DMD-associated dilated cardiomyopathy (DCM). In someembodiments, a subject is not suffering from symptoms of adystrophinopathy.

An aspect of the disclosure includes a methods involving administeringto a subject an effective amount of a complex as described herein. Insome embodiments, an effective amount of a pharmaceutical compositionthat comprises a complex comprising a muscle-targeting agent covalentlylinked to a molecular payload can be administered to a subject in needof treatment. In some embodiments, a pharmaceutical compositioncomprising a complex as described herein may be administered by asuitable route, which may include intravenous administration, e.g., as abolus or by continuous infusion over a period of time. In someembodiments, intravenous administration may be performed byintramuscular, intraperitoneal, intracerebrospinal, subcutaneous,intra-articular, intrasynovial, or intrathecal routes. In someembodiments, a pharmaceutical composition may be in solid form, aqueousform, or a liquid form. In some embodiments, an aqueous or liquid formmay be nebulized or lyophilized. In some embodiments, a nebulized orlyophilized form may be reconstituted with an aqueous or liquidsolution.

Compositions for intravenous administration may contain various carrierssuch as vegetable oils, dimethylactamide, dimethyformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injection, water soluble antibodies can be administeredby the drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload is administered via site-specific or local deliverytechniques. Examples of these techniques include implantable depotsources of the complex, local delivery catheters, site specificcarriers, direct injection, or direct application.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload is administered at an effective concentration thatconfers therapeutic effect on a subject. Effective amounts vary, asrecognized by those skilled in the art, depending on the severity of thedisease, unique characteristics of the subject being treated, e.g. age,physical conditions, health, or weight, the duration of the treatment,the nature of any concurrent therapies, the route of administration andrelated factors. These related factors are known to those in the art andmay be addressed with no more than routine experimentation. In someembodiments, an effective concentration is the maximum dose that isconsidered to be safe for the patient. In some embodiments, an effectiveconcentration will be the lowest possible concentration that providesmaximum efficacy.

Empirical considerations, e.g. the half-life of the complex in asubject, generally will contribute to determination of the concentrationof pharmaceutical composition that is used for treatment. The frequencyof administration may be empirically determined and adjusted to maximizethe efficacy of the treatment.

Generally, for administration of any of the complexes described herein,an initial candidate dosage may be about 1 to 100 mg/kg, or more,depending on the factors described above, e.g. safety or efficacy. Insome embodiments, a treatment will be administered once. In someembodiments, a treatment will be administered daily, biweekly, weekly,bimonthly, monthly, or at any time interval that provide maximumefficacy while minimizing safety risks to the subject. Generally, theefficacy and the treatment and safety risks may be monitored throughoutthe course of treatment

The efficacy of treatment may be assessed using any suitable methods. Insome embodiments, the efficacy of treatment may be assessed byevaluation of observation of symptoms associated with adystrophinopathy, e.g. muscle atrophy or muscle weakness, throughmeasures of a subject's self-reported outcomes, e.g. mobility,self-care, usual activities, pain/discomfort, and anxiety/depression, orby quality-of-life indicators, e.g. lifespan.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload described herein is administered to a subject at aneffective concentration sufficient to modulate activity or expression ofa target gene by at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90% orat least 95% relative to a control, e.g. baseline level of geneexpression prior to treatment.

In some embodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1-5, 1-10, 5-15, 10-20, 15-30, 20-40, 25-50, or more days. In someembodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In someembodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1, 2, 3, 4, 5, or 6 months.

In some embodiments, a pharmaceutical composition may comprise more thanone complex comprising a muscle-targeting agent covalently linked to amolecular payload. In some embodiments, a pharmaceutical composition mayfurther comprise any other suitable therapeutic agent for treatment of asubject, e.g. a human subject having a dystrophinopathy. In someembodiments, the other therapeutic agents may enhance or supplement theeffectiveness of the complexes described herein. In some embodiments,the other therapeutic agents may function to treat a different symptomor disease than the complexes described herein.

EXAMPLES Example 1: Targeting HPRT with Transfected AntisenseOligonucleotides

A siRNA that targets hypoxanthine phosphoribosyltransferase (HPRT) wastested in vitro for its ability to reduce expression levels of HPRT inan immortalized cell line. Briefly, Hepa 1-6 cells were transfected witheither a control siRNA (siCTRL; 100 nM) or the siRNA that targets HPRT(siHPRT; 100 nM), formulated with lipofectamine 2000. HPRT expressionlevels were evaluated 48 hours following transfection. A controlexperiment was also performed in which vehicle (phosphate-bufferedsaline) was delivered to Hepa 1-6 cells in culture and the cells weremaintained for 48 hours. As shown in FIG. 1 , it was found that the HPRTsiRNA reduced HPRT expression levels by about 90% compared withcontrols. Sequences of the siRNAs used are provided in Table 6.

TABLE 6 Sequences of siHPRT and siCTRL Sequence* SEQ ID NO:siHPRT sense strand 5′-UcCuAuGaCuGuAgAuUuUaU-(CH₂)₆NH2-3′ 147siHPRT antisense strand 5′-paUaAaAuCuAcAgUcAuAgGasAsu-3′ 148siCTRL sense strand 5′-UgUaAuAaCcAuAuCuAcCuU-(CH₂)₆NH2-3′ 149siCTRL antisense strand 5′-aAgGuAgAuAuGgUuAuUaCasAsa-3′ 150 *Lowercase-2′Ome ribose; Capital letter-2′Fluoro ribose; p-phosphate linkage;s-phosphorothioate linkage

Example 2: Targeting HPRT with a Muscle-Targeting Complex

A muscle-targeting complex was generated comprising the HPRT siRNA usedin Example 1 (siHPRT) covalently linked, via a non-cleavableN-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker, to RI7 217anti-TfR1 Fab (DTX-A-002), an anti-transferrin receptor antibody.

Briefly, the GMBS linker was dissolved in dry DMSO and coupled to the 3′end of the sense strand of siHPRT through amide bond formation underaqueous conditions. Completion of the reaction was verified by Kaisertest. Excess linker and organic solvents were removed by gel permeationchromatography. The purified, maleimide functionalized sense strand ofsiHPRT was then coupled to DTX-A-002 antibody using a Michael additionreaction.

The product of the antibody coupling reaction was then subjected to sizeexclusion chromatography (SEC) purification. antiTfR-siHPRT complexescomprising one or two siHPRT molecules covalently attached to DTX-A-002antibody were purified. Densitometry confirmed that the purified sampleof complexes had an average siHPRT to antibody ratio of 1.46. SDS-PAGEanalysis demonstrated that >90% of the purified sample of complexescomprised DTX-A-002 linked to either one or two siHPRT molecules.

Using the same methods as described above, a control IgG2a-siHPRTcomplex was generated comprising the HPRT siRNA used in Example 1(siHPRT) covalently linked via the GMBS linker to an IgG2a (Fab)antibody (DTX-A-003). Densitometry confirmed that DTX-C-001 (theIgG2a-siHPRT complex) had an average siHPRT to antibody ratio of 1.46and SDS-PAGE demonstrated that >90% of the purified sample of controlcomplexes comprised DTX-A-003 linked to either one or two siHPRTmolecules.

The antiTfR-siHPRT complex was then tested for cellular internalizationand inhibition of HPRT in cellulo. Hepa 1-6 cells, which have relativelyhigh expression levels of transferrin receptor, were incubated in thepresence of vehicle (phosphate-buffered saline), IgG2a-siHPRT (100 nM),antiTfR-siCTRL (100 nM), or antiTfR-siHPRT (100 nM), for 72 hours. Afterthe 72 hour incubation, the cells were isolated and assayed forexpression levels of HPRT (FIG. 2 ). Cells treated with theantiTfR-siHPRT demonstrated a reduction in HPRT expression by about 50%relative to the cells treated with the vehicle control and to thosetreated with the IgG2a-siHPRT complex. Meanwhile, cells treated witheither of the IgG2a-siHPRT or antiTfR-siCTRL had HPRT expression levelscomparable to the vehicle control (no reduction in HPRT expression).These data indicate that the anti-transferrin receptor antibody of theantiTfR-siHPRT enabled cellular internalization of the complex, therebyallowing the siHPRT to inhibit expression of HPRT.

Example 3: Targeting HPRT in Mouse Muscle Tissues with aMuscle-Targeting Complex

The muscle-targeting complex described in Example 2, antiTfR-siHPRT, wastested for inhibition of HPRT in mouse tissues. C57BL/6 wild-type micewere intravenously injected with a single dose of a vehicle control(phosphate-buffered saline); siHPRT (2 mg/kg of siRNA); IgG2a-siHPRT (2mg/kg of siRNA, corresponding to 9 mg/kg antibody complex); orantiTfR-siHPRT (2 mg/kg of siRNA, corresponding to 9 mg/kg antibodycomplex. Each experimental condition was replicated in four individualC57BL/6 wild-type mice. Following a three-day period after injection,the mice were euthanized and segmented into isolated tissue types.Individual tissue samples were subsequently assayed for expressionlevels of HPRT (FIGS. 3A-3B and 4A-4E).

Mice treated with the antiTfR-siHPRT complex demonstrated a reduction inHPRT expression in gastrocnemius (31% reduction; p<0.05) and heart (30%reduction; p<0.05), relative to the mice treated with the siHPRT control(FIGS. 3A-3B). Meanwhile, mice treated with the IgG2a-siHPRT complex hadHPRT expression levels comparable to the siHPRT control (little or noreduction in HPRT expression) for all assayed muscle tissue types.

Mice treated with the antiTfR-siHPRT complex demonstrated no change inHPRT expression in non-muscle tissues such as brain, liver, lung,kidney, and spleen tissues (FIGS. 4A-4E). These data indicate that theanti-transferrin receptor antibody of the antiTfR-siHPRT complex enabledcellular internalization of the complex into muscle-specific tissues inan in vivo mouse model, thereby allowing the siHPRT to inhibitexpression of HPRT. These data further demonstrate that theantiTfR-oligonucleotide complexes of the current disclosure are capableof specifically targeting muscle tissues.

Example 4: Targeting DMD with a Muscle-Targeting Complex

A muscle-targeting complex is generated comprising an antisenseoligonucleotide that targets a mutant allele of DMD (DMD ASO), for exonskipping, e.g., an oligonucleotide having a sequence as disclosed inTable 14, covalently linked, via a cathepsin cleavable linker, toDTX-A-002 (RI7 217 (Fab)), an anti-transferrin receptor antibody.

Briefly, purified Val-Cit-linker-DMD ASO is coupled to a functionalizedantibody fragment (e.g, RI7 217 (Fab) or 15G11 (Fab)) generated throughmodifying ε-amine on lysine of the antibody.

The product of the antibody coupling reaction is then subjected tohydrophobic interaction chromatography (HIC-HPLC) to purify themuscle-targeting complex. Densitometry and SDS-PAGE analysis of thepurified complex allow for determination of the average ratio ofASO-to-antibody and total purity, respectively.

Using the same methods as described above, a control complex isgenerated comprising DMD ASO covalently linked via a Val-Cit linker toan IgG2a (Fab) antibody. The purified muscle-targeting complexcomprising DTX-A-002 covalently linked to DMD ASO is then tested forcellular internalization and modulation of DMD exon skipping.Disease-relevant muscle cells that have relatively high expressionlevels of transferrin receptor, are incubated in the presence of vehiclecontrol (saline), muscle-targeting complex (100 nM), or control complex(100 nM) for 72 hours. After the 72 hour incubation, the cells areisolated and assayed for expression levels of DMD.

Example 5: Targeting DMD with a Muscle-Targeting Complex

A muscle-targeting complex (DTX-C-042) was generated comprising an PMOASO that targets exon 23 of DMD covalently linked to DTX-A-002 (RI7 217(Fab)), an anti-transferrin receptor antibody.

Briefly, aBicyclo[6.1.0]nonyne-PEG3-L-valine-L-citrulline-pentafluorophenyl ester(BCN-PEG3-Val-Cit-PFP) linker molecule was coupled to NH₂-C₆-(exon-23PMO) using an amide coupling reaction. Excess linker and organicsolvents were removed by gel permeation chromatography. The purifiedVal-Cit-linker-(exon-23 PMO) was then coupled to an azide functionalizedanti-transferrin receptor antibody (DTX-A-002) generated throughmodifying ε-amine on lysine with Azide-PEG4-PFP.

The product of the antibody coupling reaction was then purified anddensitometry confirmed that this sample of DTX-C-042 complexes had anaverage ASO to antibody ratio of 1.9.

The PMO ASO that targets exon 23 of DMD used in this Example comprises asequence consisting of GGCCAAACCUCGGCUUACCUGAAAU (SEQ ID NO: 171).

DTX-C-042 was tested for its ability to induce exon skipping of exon 23of the dystrophin gene, and to subsequently increase expression ofdystrophin protein in targeted muscles relevant to DMD in vivo. mdxmice, a DMD mouse model, were intravenously injected with a single doseof a vehicle control (saline); DTX-C-042 complex at a dose of 10 mg/kgASO; DTX-C-042 complex at a dose of 20 mg/kg ASO; or DTX-C-042 complexat a dose of 30 mg/kg ASO. Each experimental condition was replicated infour mdx mice. Four wild-type mice were also dosed with vehicle control(saline) as a control experiment.

Fourteen days after treatment, mice were euthanized and targeted muscletissues were collected. Individual muscle tissue samples weresubsequently assayed for percent skipping of exon 23 of the dystrophingene (FIG. 5 ). Additionally, dystrophin protein levels in targetedmuscles were also quantified (quantification of dystrophin in quadricepsis shown in FIGS. 6A-6B).

Mice treated with the DTX-C-042 complex demonstrated a dose-dependentincrease in the percent exon skipping of exon 23 in quadriceps,diaphragm, and heart muscles. Mice treated with the DTX-C-042 complexalso demonstrated a dose-dependent increase in the expression ofdystrophin protein in the quadriceps, with an average of >4% dystrophinprotein in mice treated with 30 mg/kg ASO equivalent of DTX-C-042.

These data demonstrate that the anti-transferrin receptor antibody ofthe antibody-ASO complex enables cellular internalization of the complexinto muscle-specific tissues in an in vivo mdx mouse model, therebyallowing the exon 23 PMO ASO to induce exon skipping of exon 23 of DMD.These data further demonstrate that the antibody-ASO complex is capableof specifically targeting muscle tissues.

Example 6: Targeting DMD with a Muscle-Targeting Complex to DemonstrateFunctional Benefit in mdx Mouse Model

Mdx mice (DMD mouse model; diseased mice) were intravenously injectedwith a single dose of a vehicle control (saline); the MDX-ASO (nakedexon 23 skipping PMO ASO, 30 mg/kg); or the DTX-C-042 complex asdescribed in Example 5 (anti-transferrin receptor antibody RI7 217 Fablinked to exon 23 skipping PMO, 30 mg/kg ASO equivalent). Eachexperimental condition was replicated in five mdx mice. Five wild-typemice (healthy mice) were also dosed with vehicle control (saline).

Two and four weeks after injection, the functional activity of alltreated mice was determined using an open field chamber experiment. Theexperiment involved three consecutive stages: (1) a 10-minute periodduring which each mouse was placed into an open field chamber; (2) a10-minute period during which each mouse was subjected to a hind limbfatigue challenge; and (3) a 10-minute period during which each mousewas placed into an open field chamber. The total horizontal distancestraveled during stages (1) and (3) were collected. The percent change inthe total distance traveled between the first and second tests. As shownin FIG. 7A, at the two week timepoint, the wild-type mice treated withsaline traveled an average of about 20% less during stage (3) relativeto stage (1); the mdx mice treated with saline traveled an average ofabout 70% less during stage (3) relative to stage (1); the mdx micetreated with MDX-ASO traveled an average of about 85% less during stage(3) relative to stage (1); and the mdx mice treated with DTX-C-042traveled an average of about 40% less during stage (3) relative to stage(1). When compared to wild-type mice treated with saline, mdx micetreated with saline performed significantly worse (as indicated by asignificant decrease in distance traveled in stage (3) relative to stage(1)). This observation is consistent with the impaired motor functionexperienced by DMD patients. mdx mice treated with naked MDX-ASO showedthe same compromised functional performance as those treated withvehicle. In contrast, the performance of mdx mice treated with DTX-C-042was not statistically different from vehicle treated wild-type mice. Asshown in FIG. 7B, at the four week timepoint, the wild-type mice treatedwith saline traveled an average of about 35% less during stage (3)relative to stage (1); the mdx mice treated with saline traveled anaverage of about 80% less during stage (3) relative to stage (1); themdx mice treated with MDX-ASO traveled an average of about 55% lessduring stage (3) relative to stage (1); and the mdx mice treated withDTX-C-042 traveled an average of about 50% less during stage (3)relative to stage (1).

Two and four weeks after injection, the activity of all treated mice wasdetermined using a cage running wheel test. Each mouse was individuallyplaced into cages with a running wheel for a 24-hour period. The 24-hourperiod involved five hours of light on followed by thirteen hours ofdarkness, and ending with six hours of light. The total distancetraveled (in meters, m) by each mouse on the running wheel wascontinuously collected throughout the 24-hour period and subsequentlybinned into discrete one-hour increments. As shown in FIG. 7C, at thetwo week timepoint, the distance traveled by the mdx mice treated withDTX-C-042 was higher than the distance traveled by the mdx mice treatedwith MDX-ASO or with saline, and approached the distance traveled by thewild-type mice at certain times. As shown in FIG. 7D, at the four weektimepoint, the distance traveled by the mdx mice treated with DTX-C-042complex closely mirrored the total distance traveled by the wild-typemice treated with saline during the dark period (i.e., when mice areactive). This is in contrast to the mdx mice treated with either salineor MDX-ASO, which traveled considerably shorter distances during thedark period.

All mice in this Example were further tested for creatine kinaseactivity levels two weeks and four weeks after injection. Wild-type micedo not secrete large amounts of creatine kinase from muscle tissues.Conversely, mdx mice (having diseased muscle tissues) do secrete highlevels of creatine kinase, which can be observed by determination ofcreatine kinase enzymatic activities. As shown in FIG. 7E, the mdx micethat were treated with saline had approximately 9- and 10-fold morecreatine kinase enzymatic activity relative to wild-type mice treatedwith saline after two and four weeks, respectively. Dosing with nakedASO provided no significant benefit to the mdx mice. However, dosing mdxmice with DTX-C-042 complex did provide a statistically significantreduction in levels of creatine kinase activity after both two and fourweeks.

These surprising results show that the antibody-ASO complex is capableof providing functional benefits to mice suffering from a DMD phenotype(mdx mice), such that these mice have phenotypic indicators resemblinghealthy (wild-type) mice. The performance of the antibody-ASO complexrelative to the naked PMO (MDX-ASO) demonstrates that theanti-transferrin receptor antibody of the antibody-ASO complex isresponsible for providing the functional benefits shown in this Example.

Example 7: Binding Affinity of Selected Anti-TfR1 Antibodies to HumanTfR1

Selected anti-TfR1 antibodies were tested for their binding affinity tohuman TfR1 for measurement of Ka (association rate constant), Kd(dissociation rate constant), and K_(D) (affinity). Two known anti-TfR1antibodies were used as control, 15G11 and OKT9. The binding experimentwas performed on Carterra LSA at 25° C. An anti-mouse IgG and anti-humanIgG antibody “lawn” was prepared on a HC30M chip by amine coupling. TheIgGs were captured on the chip. Dilution series of hTfR1, cyTfR1, andhTfR2 were injected to the chip for binding (starting from 1000 nM, 1:3dilution, 8 concentrations).

Binding data were referenced by subtracting the responses from a bufferanalyte injection and globally fitting to a 1:1 Langmuir binding modelfor estimate of Ka (association rate constant), Kd (dissociation rateconstant), and K_(D) (affinity) using the Carterra™ Kinetics software.5-6 concentrations were used for curve fitting.

The result showed that the mouse mAbs demonstrated binding to hTfR1 withK_(D) values ranging from 13 pM to 50 nM. A majority of the mouse mAbshad K_(D) values in the single digit nanomolar to sub-nanomolar range.The tested mouse mAbs showed cross-reactive binding to cyTfR1 with K_(D)values ranging from 16 pM to 22 nM.

Ka, Kd, and K_(D) values of anti-TfR1 antibodies are provided in Table7.

TABLE 7 Ka, Kd, and K_(D) values of anti-TfR1 antibodies Name K_(D) (M)Ka (M) Kd (M) ctrl-15G11 2.83E−10 3.70E+05 1.04E−04 ctrl-OKT9 mIgG5.36E−10 7.74E+05 4.15E−04 3-A04 4.36E−10 4.47E+05 1.95E−04 3-M127.68E−10 1.66E+05 1.27E−04 5-H12 2.08E−07 6.67E+04 1.39E−02

Example 8: Conjugation of Anti-TfR1 Antibodies with Oligonucleotides

Complexes containing an anti-TfR1 antibody covalently conjugated to atool oligo (ASO300) were generated. First, Fab fragments of anti-TfRantibody clones 3-A4, 3-M12, and 5-H12 were prepared by cutting themouse monoclonal antibodies with an enzyme in or below the hinge regionof the full IgG followed by partial reduction. The Fabs were confirmedto be comparable to mAbs in avidity or affinity.

Muscle-targeting complexes were generated by covalently linking theanti-TfR mAbs to the ASO300 via a cathepsin cleavable linker. Briefly, aBicyclo[6.1.0]nonyne-PEG3-L-valine-L-citrulline-pentafluorophenyl ester(BCN-PEG3-Val-Cit-PFP) linker molecule was coupled to the ASO300 througha carbamate bond. Excess linker and organic solvents were removed bytangential flow filtration (TFF). The purified Val-Cit-linker-ASO wasthen coupled to an azide functionalized anti-transferrin receptorantibody generated through modifying ε-amine on lysine withAzide-PEG4-PFP. A positive control muscle-targeting complex was alsogenerated using 15G11.

The product of the antibody coupling reaction was then subjected to twopurification methods to remove free antibody and free payload.Concentrations of the conjugates were determined by either Nanodrop A280or BCA protein assay (for antibody) and Quant-It Ribogreen assay (forpayload). Corresponding drug-antibody ratios (DARs) were calculated.DARs ranged between 0.8 and 2.0, and were standardized so that allsamples receive equal amounts of payload.

The purified complexes were then tested for cellular internalization andinhibition of the target gene, DMPK. Non-human primate (NHP) or DM1(donated by DM1 patients) cells were grown in 96-well plates anddifferentiated into myotubes for 7 days. Cells were then treated withescalating concentrations (0.5 nM, 5 nM, 50 nM) of each complex for 72hours. Cells were harvested, RNA was isolated, and reverse transcriptionwas performed to generate cDNA. qPCR was performed using TaqMan kitsspecific for Ppib (control) and DMPK on the QuantStudio 7. The relativeamounts of remaining DMPK transcript in treated vs non-treated cellswere calculated and the results are shown in Table 12.

The results demonstrated that the anti-TfR1 antibodies are able totarget muscle cells, be internalized by the muscle cells with themolecular payload (the tool oligo ASO300), and that the molecularpayload is able to target and knockdown the target gene (DMPK).Knockdown activity of a complex comprising the anti-TfR1 antibodyconjugated to a molecular payload (e.g., an oligonucleotide) targetingDMD may be tested using the same assay as described herein, e.g., byusing any one of the oligonucleotides described in Table 14, provided byany one of SEQ ID NO: 437-1241, or complementary to any one of SEQ IDNO: 1242-2046.

TABLE 12 Binding Affinity of anti-TfR1 Antibodies and Efficacy ofConjugates % knockdown of % knockdown of DMPK in cells huTfR1 cyTfR1DMPK in NHP from human DM1 Avg K_(D) Avg K_(D) cells using patientsusing (M) (M) Antibody-DMPK Antibody-DMPK Clone Name (antibody alone)(antibody alone) ASO conjugate ASO conjugate 15G11 (control)  8.0E−10 1.0E−09 36 46 3-A4 4.36E−10 2.32E−09 77 70 3-M12 7.68E−10 5.18E−09 7752 5-H12 2.02316E−07   1.20E−08 88 57

Interestingly, the DMPK knockdown results showed a lack of correlationbetween the binding affinity of the anti-TfR to transferrin receptor andefficacy in delivering a DMPK ASO to cells for DMPK knockdown.Surprisingly, the anti-TfR antibodies provided herein (e.g., at least3-A4, 3-M12, and 5-H12) demonstrated superior activity in delivering apayload (e.g., DMPK ASO) to the target cells and achieving thebiological effect of the molecular payload (e.g., DMPK knockdown) ineither cyno cells or human DM1 patient cells, compared to the controlantibody 15G11, despite the comparable binding affinity (or, in certaininstances, such as 5-H12, lower binding affinity) to human or cynotransferrin receptor between these antibodies and the control antibody15G11.

Top attributes such as high huTfR1 affinity, >50% knockdown of DMPK inNHP and DM1 patient cell line, identified epitope binding with 3 uniquesequences, low/no predicted PTM sites, and good expression andconjugation efficiency led to the selection of the top 3 clones forhumanization, 3-A4, 3-M12, and 5-H12.

Example 9: Humanized Anti-TfR1 Antibodies

The anti-TfR antibodies shown in Table 2 were subjected to humanizationand mutagenesis to reduce manufacturability liabilities. The humanizedvariants were screened and tested for their binding properties andbiological actives. Humanized variants of anti-TfR1 heavy and lightchain variable regions (5 variants each) were designed using CompositeHuman Technology. Genes encoding Fabs having these heavy and light chainvariable regions were synthesized, and vectors were constructed toexpress each humanized heavy and light chain variant. Subsequently, eachvector was expressed on a small scale and the resultant humanizedanti-TfR1 Fabs were analyzed. Humanized Fabs were selected for furthertesting based upon several criteria including Biocore assays of antibodyaffinity for the target antigen, relative expression, percent homologyto human germline sequence, and the number of MHC class II predicted Tcell epitopes (determined using iTope™ MCH class II in silico analysis).

Potential liabilities were identified within the parental sequence ofsome antibodies by introducing amino acid substitutions in the heavychain and light chain variable regions. These substitutions were chosenbased on relative expression levels, iTope™ score and relative K_(D)from Biacore single cycle kinetics analysis. The humanized variants weretested and variants were selected initially based upon affinity for thetarget antigen. Subsequently, the selected humanized Fabs were furtherscreened based on a series of biophysical assessments of stability andsusceptibility to aggregation and degradation of each analyzed variant,shown in Table 16 and Table 17. The selected Fabs were analyzed fortheir properties binding to TfR1 by kinetic analysis. The results ofthese analyses are shown in Table 13. For conjugates shown in Table 16and Table 17, the selected humanized Fabs were conjugated to aDMPK-targeting oligonucleotide ASO300. The selected Fabs are thermallystable, as indicated by the comparable binding affinity to human andcyno TfR1 after been exposed to high temperature (40° C.) for 9 days,compared to before the exposure (see Table 13).

TABLE 16 Biophysical assessment data for humanized anti-TfR Fabs Variant3M12 3M12 3M12 3M12 3A4 (VH3- Criteria (VH3/Vk2) (VH3/Vk3) (VH4/Vk2)(VH4/Vk3) N54T/Vk4) Binding Affinity 395 pM 345 pM 396 pM 341 pM 3.09 nM(Biacore d0) Binding Affinity 567 pM 515 pM 510 pM 486 pM 3.01 nM(Biacore d25) Fab binding 0.8 nM/ 0.6 nM/ 0.4 nM/ 0.5 nM/ 2.6 nM/affinity ELISA 9.9 nM 4.7 nM 1.4 nM 2.2 nM 156 nM* (human/cyno TfR1)Conjugate binding 2.2 nM/ N/A N/A 1.7 nM/ 2.8 nM/ affinity ELISA 2.9 nM2.1 nM 4.7 nM (human/cyno TfR1) . . . Variant 3A4 (VH3- 3A4 5H12 (VH5-5H12 (VH5- 5H12 (VH4- Criteria N54S/Vk4) (VH3/Vk4) C33Y/Vk3) C33D/Vk4)C33Y/Vk4) Binding Affinity 1.34 nM  1.5 nM  627 pM  991 pM  626 pM(Biacore d0) Binding Affinity 1.39 nM 1.35 nM 1.07 nM 3.01 nM 1.33 nM(Biacore d25) Fab binding 1.6 nM/ 1.5 nM/ 6.3 nM/ 6.0 nM/ 2.8 nM/affinity ELISA 398 nM* 122 nM* 2.1 nM 3.5 nM 3.3 nM (human/cyno TfR1)Conjugate binding 2.9 nM/ 2.8 nM/ 33.4 nM/ 110 nM/ 23.7 nM/ affinityELISA 7.8 nM 7.6 nM 2.3 nM 10.2 nM 3.3 nM (human/cyno TfR1) *Regainscyno binding after conjugation;

TABLE 17 Thermal Stability for humanized anti-TfR Fabs and conjugatesVariant 3M12 3M12 3M12 3M12 3A4 (VH3- Criteria (VH3/Vk2) (VH3/Vk3)(VH4/Vk2) (VH4/Vk3) N54T/Vk4) Binding affinity 0.8 0.6 0.4 0.5 2.6 hTfR1d0 (nM) Binding affinity 0.98 1.49 0.50 0.28 0.40 hTfR1 d9 (nM) Bindingaffinity 9.9 4.7 1.4 2.2 156 cyno TfR1 d0 (nM) Binding affinity 19.5115.58 5.01 16.40 127.50 cyno TfR1 d9 (nM) DMPK oligo 1.14 N/A N/A 1.182.22 conjugate binding to hTfR1 (nM) DMPK oligo 2.26 N/A N/A 1.85 5.12conjugate binding to cyno TfR1 (nM) . . . Variant 3A4 (VH3- 3A4 5H12(VH5- 5H12 (VH5- 5H12 (VH4- Criteria N54S/Vk4) (VH3/Vk4) C33Y/Vk3)C33D/Vk4) C33Y/Vk4) Binding affinity 1.6 1.5 6.3 6 2.8 hTfR1 d0 (nM)Binding affinity 0.65 0.46 71.90 92.34 1731.00 hTfR1 d9 (nM) Bindingaffinity 398 122 2.1 3.5 3.3 cyno TfR1 d0 (nM) Binding affinity 248.30878.40 0.69 0.63 0.26 cyno TfR1 d9 (nM) DMPK oligo 2.71 2.837 N/A 110.513.9 conjugate binding to hTfR1 (nM) DMPK oligo 4.1 7.594 N/A 10.18 13.9conjugate binding to cyno TfR1 (nM)

TABLE 13 Kinetic analysis of humanized anti-TfR Fabs binding to TfR1Humanized anti-TfR Fabs k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) R_(MAX) Chi²(RU²) 3A4 (VH3/Vk4) 7.65E+10 1.15E+02 1.50E−09 48.0 0.776 3A4(VH3-N54S/Vk4) 4.90E+10 6.56E+01 1.34E−09 49.4 0.622 3A4 (VH3-N54T/Vk4)2.28E+05 7.05E−04 3.09E−09 61.1 1.650 3M12 (VH3/Vk2) 2.64E+05 1.04E−043.95E−10 78.4 0.037 3M12 (VH3/Vk3) 2.42E+05 8.34E−05 3.45E−10 91.1 0.0253M12 (VH4/Vk2) 2.52E+05 9.98E−05 3.96E−10 74.8 0.024 3M12 (VH4/Vk3)2.52E+05 8.61E−05 3.41E−10 82.4 0.030 5H12 (VH5-C33D/Vk4) 6.78E+056.72E−04 9.91E−10 49.3 0.093 5H12 (VH5-C33Y/Vk3) 1.95E+05 1.22E−046.27E−10 68.5 0.021 5H12 (VH5-C33Y/Vk4) 1.86E+05 1.17E−04 6.26E−10 75.20.026

Binding of Humanized Anti-TfR1 Fabs to TfR1 (ELISA)

To measure binding of humanized anti-TfR antibodies to TfR1, ELISAs wereconducted. High binding, black, flat bottom, 96 well plates (Corning#3925) were first coated with 100 μL/well of recombinant huTfR1 at 1μg/mL in PBS and incubated at 4° C. overnight. Wells were emptied andresidual liquid was removed. Blocking was conducted by adding 200 μL of1% BSA (w/w) in PBS to each well. Blocking was allowed to proceed for 2hours at room temperature on a shaker at 300 rpm. After blocking, liquidwas removed and wells were washed three times with 300 μL of TBST.Anti-TfR1 antibodies were then added in 0.5% BSA/TBST in triplicate inan 8 point serial dilution (dilution range 5 μg/mL-5 ng/mL). A positivecontrol and isotype controls were also included on the ELISA plate. Theplate was incubated at room temperature on an orbital shaker for 60minutes at 300 rpm, and the plate was washed three times with 300 μL ofTBST. Anti-(H+L)IgG-A488 (1:500) (Invitrogen #A11013) was diluted in0.5% BSA in TBST, and 100 μL was added to each well. The plate was thenallowed to incubate at room temperature for 60 minutes at 300 rpm onorbital shaker. The liquid was removed and the plate was washed fourtimes with 300 μL of TBST. Absorbance was then measured at 495 nmexcitation and 50 nm emission (with a 15 nm bandwidth) on a platereader. Data was recorded and analyzed for EC₅₀. The data for binding tohuman TfR1 (hTfR1) for the humanized 3M12, 3A4 and 5H12 Fabs are shownin FIGS. 9A, 9C, and 9E, respectively. ELISA measurements were conductedusing cynomolgus monkey (Macaca fascicularis) TfR1 (cTfR1) according tothe same protocol described above for hTfR1, and results are shown inFIGS. 9B, 9D, and 9F.

Results of these two sets of ELISA analyses for binding of the humanizedanti-TfR Fabs to hTfR1 and cTfR1 demonstrate that humanized 3M12 Fabsshow consistent binding to both hTfR1 and cTfR1, and that humanized 3A4Fabs show decreased binding to cTfR1 relative to hTfR1.

Antibody-oligonucleotide conjugates were prepared using six humanizedanti-TfR Fabs, each of which were conjugated to a DMPK targetingoligonucleotide ASO300. Conjugation efficiency and down-streampurification were characterized, and various properties of the productconjugates were measured. The results demonstrate that conjugationefficiency was robust across all 10 variants tested, and that thepurification process (hydrophobic interaction chromatography followed byhydroxyapatite resin chromatography) were effective. The purifiedconjugates showed a >97% purity as analyzed by size exclusionchromatography.

Several humanized Fabs were tested in cellular uptake experiments toevaluate TfR1-mediated internalization. To measure such cellular uptakemediated by antibodies, humanized anti-TfR Fab conjugates were labeledwith Cypher5e, a pH-sensitive dye. Rhabdomyosarcoma (RD) cells weretreated for 4 hours with 100 nM of the conjugates, trypsinized, washedtwice, and analyzed by flow cytometry. Mean Cypher5e fluorescence(representing uptake) was calculated using Attune N×T software. As shownin FIG. 10 , the humanized anti-TfR Fabs show similar or greaterendosomal uptake compared to a positive control anti-TfR1 Fab. Similarinternalization efficiencies were observed for different oligonucleotidepayloads. An anti-mouse TfR antibody was used as the negative control.Cold (non-internalizing) conditions abrogated the fluorescence signal ofthe positive control antibody-conjugate (data not shown), indicatingthat the positive signal in the positive control and humanized anti-TfRFab-conjugates is due to internalization of the Fab-conjugates.Similarly, an oligonucleotide that induces DMD exon skipping can also beconjugated the humanized anti-TfR Fabs for cellular uptake by musclecells.

Conjugates of six humanized anti-TfR Fabs of were also tested forbinding to hTfR1 and cTfR1 by ELISA, and compared to the unconjugatedforms of the humanized Fabs. Results demonstrate that humanized 3M12 and5H12 Fabs maintain similar levels of hTfR1 and cTfR1 binding afterconjugation relative to their unconjugated forms (3M12, FIGS. 11A and11B; 5H12, FIGS. 11E and 11F). Interestingly, 3A4 clones show improvedbinding to cTfR1 after conjugation relative to their unconjugated forms(FIGS. 11C and 11D).

As used in this Example, the term ‘unconjugated’ indicates that theantibody was not conjugated to an oligonucleotide.

Example 10. Knockdown of DMPK mRNA Level Facilitated byAntibody-Oligonucleotide Conjugates In Vitro

Conjugates containing humanized anti-TfR Fabs 3M12(VH3/Vk2), 3M-12(VH4/Vk3), and 3A4(VH3-N54S/Vk4) were conjugated to a DMPK-targetingoligonucleotide ASO300 and were tested in rhabdomyosarcoma (RD) cellsfor knockdown of DMPK transcript expression. Antibodies were conjugatedto ASO300 via the linker shown in Formula (C).

RD cells were cultured in a growth medium of DMEM with glutamine,supplemented with 10% FBS and penicillin/streptomycin until nearlyconfluent. Cells were then seeded into a 96 well plate at 20K cells perwell and were allowed to recover for 24 hours. Cells were then treatedwith the conjugates for 3 days. Total RNA was collected from cells, cDNAwas synthesized and DMPK expression was measured by qPCR.

Results in FIG. 12 show that DMPK expression level was reduced in cellstreated with each indicated conjugate, relative to expression inPBS-treated cells, indicating that the humanized anti-TfR Fabs are ableto mediate the uptake of the DMPK-targeting oligonucleotide by the RDcells and that the internalized DMPK-targeting oligonucleotide areeffective in knocking down DMPK mRNA level.

Similarly, an oligonucleotide that induces DMD exon skipping can also beconjugated the humanized anti-TfR Fabs for delivery to muscle cells andinducing DMD exon skipping in muscle cells.

Example 11. Anti-TfR-Oligonucleotide Conjugate Treatment IncreasedDystrophin Expression in mdx Mouse Model of DMD

To test the effects of another oligonucleotide that induces DMD exonskipping in vivo, an oligonucleotide (PMO) that induces exon 23 skippingwas administered as naked oligonucleotide or in conjugate with ananti-mouse TfR antibody to the mdx mouse model of DMD. Dystrophinexpression was measured. The exon skipping promoted by the conjugateresulted in dose-dependent production of dystrophin protein asillustrated by western blot (FIG. 14 ) and quantified in FIG. 15 .Alpha-actin was used as a loading control.

A single dose of the exon 23-conjugate administered in the mdx mousealso restored dystrophin expression to the muscle cell membrane inaddition to increasing overall dystrophin levels, as shown in FIG. 16 .Immunofluorescence staining of dystrophin in quadricep musclesdemonstrated that mdx mice treated with the conjugated had higher levelsof dystrophin in their quadriceps than mice treated with nakedoligonucleotide or saline.

Example 12. Oligonucleotide-Mediated Exon Skipping in DMD Myotubes

Promoting the skipping of specific DMD exons in the nucleus could allowmuscle cells to create more complete, functional dystrophin protein. Anoligonucleotide (PMO) that induces skipping of DMD exon 51 wasconjugated to an anti-TfR1 Fab and the conjugated was tested in humanDMD myotubes with a mutation amenable to Exon 51 skipping. Treatmentwith the conjugate resulted in a 50% increase in exon skipping ascompared to a 25% increase in exon skipping following treatment with anequimolar dose of the naked oligonucleotide (p-value=0.001), as shown inFIG. 13 . Similar results were observed in the mdx mouse model of DMD,such as those shown in FIG. 5 .

Example 13. Serum Stability of the Linker Linking the Anti-TfR Antibodyand the Molecular Payload

Oligonucleotides which were linked to antibodies in examples wereconjugated via a cleavable linker shown in Formula (C). It is importantthat the linker maintain stability in serum and provide release kineticsthat favor sufficient payload accumulation in the targeted muscle cell.This serum stability is important for systemic intravenousadministration, stability of the conjugated oligonucleotide in thebloodstream, delivery to muscle tissue and internalization of thetherapeutic payload in the muscle cells. The linker has been confirmedto facilitate precise conjugation of multiple types of payloads(including ASOs, siRNAs and PMOs) to Fabs. This flexibility enabledrational selection of the appropriate type of payload to address thegenetic basis of each muscle disease. Additionally, the linker andconjugation chemistry allowed the optimization of the ratio of payloadmolecules attached to each Fab for each type of payload, and enabledrapid design, production and screening of molecules to enable use invarious muscle disease applications.

FIG. 8 shows serum stability of the linker in vivo, which was comparableacross multiple species over the course of 72 hours after intravenousdosing. At least 75% stability was measured in each case at 72 hoursafter dosing.

Example 14. Exon-Skipping Activity of Anti-TfR Conjugates in DMD PatientMyotubes

In this study, the exon-skipping activities of anti-TfR conjugatescontaining an anti-TfR Fab (3M12 VH3/Vκ2, 3M12 VH4/Vκ3, and 3A4 VH3N54S/Vκ4) conjugated to a DMD exon 51-skipping oligonucleotide wereevaluated. Immortalized human myoblasts bearing an exon 52 deletion werethawed and seeded at a density of 1 e6 cell/flask in Promocell SkeletalCell Growth Media (with 5% FBS and 1× Pen-Strep) and allowed to grow toconfluency. Once confluent, cells were trypsinized and pelleted viacentrifugation and resuspended in fresh Promocell Skeletal Cell GrowthMedia. The cell number was counted and cells were seeded intoMatrigel-coated 96-well plates at a density of 50k cells/well. Cellswere allowed to recover for 24 hours. Cells were induced todifferentiate by aspirating the growth media and replacing withdifferentiation media with no serum. Cells were then treated withconjugated or unconjugated DMD exon skipping oligonucleotide at 10 μM.Cells were incubated with test articles for ten days then total RNA washarvested from the 96 well plates. cDNA synthesis was performed on 75 ngof total RNA, and mutation specific PCRs were performed to evaluate thedegree of exon 51 skipping in each cell type. Mutation-specific PCRproducts were run on a 4% agarose gel and visualized using SYBR gold.Densitometry was used to calculate the relative amounts of the skippedand unskipped amplicon and exon skipping was determined as a ratio ofthe Exon 51 skipped amplicon divided by the total amount of ampliconpresent:

${\%{{Exon}{Skipping}}} = {\frac{{Skipped}{Amplicon}}{\left( {{{Skipped}{Amplicon}} + {{Unskipped}{Amplicon}}} \right)}*100}$

The results demonstrate that the conjugates with either 3M12 VH3/Vκ2 or3M12 VH4/Vκ3 Fab covalently linked to the DMD exon 51-skippingoligonucleotide resulted in enhanced exon skipping compared to theunconjugated DMD exon skipping oligonucleotide in patient myotubes (FIG.17 ).

As used in this Example, the term ‘unconjugated’ indicates that theoligonucleotide was not conjugated to an antibody.

Example 15. Characterization of Binding Activities of Anti-TfR Fab 3M12VH4/Vk3

In vitro studies were performed to test the specificity of anti-TfR Fab3M12 VH4/Vκ3 for human and cynomolgus monkey TfR1 binding and to confirmits selectivity for human TfR1 vs TfR2. The binding affinity of anti-TfRFab 3M12 VH4/Vκ3 to TfR1 from various species was determined using anenzyme-linked immunosorbent assay (ELISA). Serial dilutions of the Fabwere added to plates precoated with recombinant human, cynomolgusmonkey, mouse, or rat TfR1. After a short incubation, binding of the Fabwas quantified by addition of a fluorescently tagged anti-(H+L) IgGsecondary antibody and measurement of fluorescence intensity at 495 nmexcitation and 520 nm emission. The Fab showed strong binding affinityto human and cynomolgus monkey TfR1, and no detectable binding of mouseor rat TfR1 was observed (FIG. 18 ). Surface plasmon resonance (SPR)measurements were also conducted, and results are shown in Table 18. TheK_(d) of the Fab against the human TfR1 receptor was calculated to be7.68×10⁻¹⁰ M and against the cynomolgus monkey TfR1 receptor wascalculated to be 5.18×10⁻⁹M.

TABLE 18 Kinetic analysis of anti-TfR Fab 3M12 VH4/Vk3 binding to humanand cynomolgus monkey TfR1 or human TfR2, measured using surface plasmonresonance Anti-TfR Fab 3M12 VH4/Vk3 Target K_(d) (M) k_(a) (M⁻¹ s⁻¹)k_(d) (s⁻¹) R_(max) R_(es) SD Human 7.68E−10 1.66E+05 1.27E−04 1.11E+023.45E+00 TfR1 Cyno 5.18E−09 9.19E+04 4.76E−04 1.87E+02 6.24E+00 TfR1Human ND ND ND ND ND TfR2 ND = No detectable binding by SPR (10 pM-100uM)

To test for cross-reactivity of anti-TfR Fab 3M12 VH4/Vk3 to human TfR2,an ELISA was performed. Recombinant human TfR2 protein was platedovernight at 2 μg/mL and was blocked for 1 hour with 1% bovine serumalbumin (BSA) in PBS. Serial dilutions of the Fab or a positive controlanti-TfR2 antibody were added in 0.5% BSA/TBST for 1 hour. Afterwashing, anti-(H+L) IgG-A488 (Invitrogen #MA5-25932) fluorescentsecondary antibody was added at a 1:500 dilution in 0.5% BSA/TBST andthe plate was incubated for 1 hour. Relative fluorescence was measuredusing a Biotek Synergy plate reader at 495 nm excitation and 520 nmemission. No binding of anti-TfR Fab 3M12 VH4/Vk3 to hTfR2 was observed(FIG. 19 ).

Example 16. Serum Stability of Anti-TfR Fab-ASO Conjugate

Anti-TfR Fab VH4/Vk3 was conjugated to a control antisenseoligonucleotide (ASO) via a linker as shown in Formula (C) and theresulting conjugate was tested for stability of the linker conjugatingthe Fab to the ASO. Serum stability was measured by incubatingfluorescently labeled conjugate in PBS or in rat, mouse, cynomolgusmonkey, or human serum and measuring relative fluorescence intensityover time, with higher fluorescence indicating more conjugate remainingintact. FIG. 20 shows serum stability was similar across multiplespecies and remained high after 72 hours.

Example 17. Exon Skipping Activity of Anti-TfR Fab-ASO Conjugate In Vivoin Cynomolgus Monkeys

Anti-TfR Fab 3M12 VH4/Vk3 was conjugated to a dystrophin (DMD) exon51-skipping antisense oligonucleotide (ASO) targeting an ESE as setforth in SEQ ID NO: 419. The exon 51 skipping oligonucleotide is aphosphorodiamidate morpholino oligomer (PMO) of 30 nucleotides inlength. The exon skipping activity of the conjugate was tested in vivoin healthy non-human primates. Naïve male cynomolgus monkeys (n=4-5 pergroup) were administered two doses of vehicle, 30 mg/kg ASO alone, or122 mg/kg conjugate (30 mg/kg ASO equivalent) via intravenous infusionon days 1 and 8. Animals were sacrificed and tissues harvested either 2weeks or 4 weeks after the first dose was administered. Total RNA wascollected from tissue samples using a Promega Maxwell® RSC instrumentand cDNA synthesis was performed using qScript cDNA SuperMix. Assessmentof exon 51 skipping was performed using end-point PCR.

Capillary electrophoresis of the PCR products was used to assess exonskipping, and % exon 51 skipping was calculated using the followingformula:

${\%{{Exon}{Skipping}}} = {\frac{{Molarity}{of}{Skipped}{Band}}{{{Molarity}{of}{Skipped}{Band}} + {{Molarity}{of}{Unskipped}{Band}}}*100.}$

Calculated exon 51 skipping results are shown in Table 19.

TABLE 19 Exon 51 skipping of dystrophin in cynomolgus monkey dystrophinTime 2 weeks 4 weeks Group ASO Conju- ASO Conju- Vehicle alone^(a) gatealone^(a) gate Conjugate dose^(b) 0 n/a 122 n/a 122 ASO alone Dose^(c) 030  30 30  30 Quadriceps ^(d) 0.00 1.216 4.906 0.840 1.708 (0.00)(1.083) (3.131) (1.169) (1.395) Diaphragm ^(d) 0.00 1.891 7.315 0.7179.225 (0.00) (2.911) (1.532) (1.315) (4.696) Heart ^(d) 0.00 0.043 3.420.00 4.525 (0.00) (0.096) (1.192) (0.00) (1.400) Biceps ^(d) 0.00 0.6073.129 1.214 4.863 (0.00) (0.615) (0.912) (1.441) (3.881) Tibialisanterior ^(d) 0.00 0.699 1.042 0.384 0.816 (0.00) (0.997) (0.685)(0.615) (0.915) Gastrocnemius ^(d) 0.00 0.388 2.424 0.00 5.393 (0.00)(0.573) (2.329) (0.00) (2.695) ^(a)ASO = antisense oligonucleotide.^(b)Conjugate doses are listed as mg/kg of anti-TfR Fab 3M12 VH4/Vk3-ASOconjugate. ^(c)ASO doses are listed as mg/kg ASO equivalent of theanti-TfR Fab 3M12 VH4/Vk3-ASO dose. ^(d) Exon skipping values are mean %exon 51 skipping with standard deviations (n = 5) in parentheses.

Tissue ASO accumulation was also quantified using a hybridization ELISAwith a probe complementary to the ASO sequence. A standard curve wasgenerated and ASO levels (in ng/g) were derived from a linear regressionof the standard curve. The ASO was distributed to all tissues evaluatedat a higher level following the administration of the anti-TfR FabVH4/Vk3-ASO conjugate as compared to the administration of unconjugatedASO. Intravenous administration of unconjugated ASO resulted in levelsof ASO that were close to background levels in all tissues evaluated at2 and 4 weeks after the first does was administered. Administration ofanti-TfR Fab VH4/Vk3-ASO conjugate resulted in distribution of ASOthrough the tissues evaluated with a rank order ofheart>diaphragm>bicep>quadriceps>gastrocnemious>tibialis anterior 2weeks after first dosing. The duration of tissue concentration was alsoassessed. Concentrations of the ASO in quadriceps, bicep and diaphragmdecreased by less than 50% over the time period evaluated (2 to 4weeks), while levels of ASO in the heart, tibialis anterior, andgastrocnemius remained virtually unchanged (Table 20).

As used in this Example, the term ‘unconjugated’ indicates that theoligonucleotide was not conjugated to an antibody.

TABLE 20 Tissue distribution of DMD exon 51 skipping ASO in cynomolgusmonkeys Time 2 weeks 4 weeks Group ASO Conju- ASO Conju- Vehiclealone^(a) gate alone^(a) gate Conjugate Dose^(b) 0 n/a 122 n/a 122 ASOalone Dose^(c) 0 30  30 30  30 Quadriceps ^(d) 0 696.8 2436 197 682(59.05) (868.15) (954.0) (134) (281) Diaphragm ^(d) 0± 580.02 6750 603131 (144.3) (360.11) (2256) (120) (1618) Heart ^(d) 0 1449 27138 94330410 (396.03) (1337) (6315) (1803) (9247) Biceps ^(d) 0 615.63 2840 1301326 (69.58) (335.17) (980.31) (80) (623) Tibialis anterior ^(d) 0564.71 1591 169 1087 (76.31) (327.88) (253.50) (110) (514) Gastrocnemius^(d) 0 705.47 2096 170 1265 (41.15) (863.75) (474.04) (69) (272) ^(a)ASO= Antisense oligonucleotide. ^(b)Conjugate doses are listed as mg/kg ofanti-TfR Fab 3M12 VH4/Vk3-ASO conjugate. ^(c)ASO doses are listed asmg/kg ASO or ASO equivalent of the anti-TfR Fab 3M12 VH4/Vk3-ASOconjugate dose. ^(d) ASO values are mean concentrations of ASO in tissueas ng/g with standard deviations (n = 5) in parentheses.

Example 18. Effect of Conjugates Containing an Anti-TfR1 Fab Conjugatedto an Oligonucleotide that Induces DMD Exon 23 Skipping on BiomarkerExpression and Muscle Function in mdx Mice

The objective of this study was to determine the effect of a single doseof anti-mouse TfR Fab conjugated to an antisense oligonucleotide(Ab-ASO) or of a single dose of the same naked ASO on dystrophinexpression and muscle function in mdx mice. The complex used in thisexample was DTX-C-042 as described in Example 5.

Seven-week-old male mdx homozygous mice were allocated randomly to eachof eight treatment groups. The mice were administered via tail vein asingled dose of ASO at 30 mg/kg, Ab-ASO at a dose equivalent to 30 mg/kgof ASO, or saline. Tissues were harvested and analyzed 2 weeks or 4weeks following administration.

Measurement of exon 23 skipping in muscles: Quantification of exon 23skipping was performed using a single-step RT-PCR reaction using theSuperScript® III (Thermo Fisher) with 75 ng total RNA input. The PCRprimers used were 5′-CACATCTTTGATGGTGTGAGG (forward) (SEQ ID NO: 2264)and 5′-CAACTTCAGCCATCCATTTCTG (reverse) (SEQ ID NO: 2253). Capillaryelectrophoresis was used to quantitate the skipped and unskipped bandsin the skeletal muscles of interest using the following equation:

${\%{{Exon}23{Skipping}}} = {\frac{{Skipped}{Band}}{{{Skipped}{Band}} + {{Unskipped}{Band}}} \times 100.}$

The results demonstrate that a single administration of anti-TfRFab-oligonucleotide conjugate (Ab-ASO) facilitated significant increasesin skipping of exon 23 in quadriceps (FIG. 21A), heart (FIG. 21B), anddiaphragm (FIG. 21C) of mdx mice. By contrast, little or no exon 23skipping was observed in the same muscle tissues in wild-type (WT) miceor in mdx mice treated with saline or naked ASO.

Measurement of dystrophin protein in muscles: Muscle tissue samplestaken from the quadriceps were homogenized and protein concentrationswere measured by BCA assay. Total protein (25 μg) was loaded onto a 3%to 8% Tris-acetate protein gel and run at 150 V for 1 hour. The gel wasthen transferred to a polyvinylidene difluoride membrane, and themembrane was cut, and the portion containing dystrophin was incubatedovernight in anti-dystrophin antibody (Abcam catalog #15277) at 4° C.,followed by goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate(Bio-Rad) for 30 minutes at room temperature. As a control, theremaining portion of the blot was incubated overnight withanti-alpha-actinin antibody (Abcam catalog #9465) at 4° C., followed bygoat anti-mouse IgG (H+L) horseradish peroxidase conjugate (Bio-Rad) for15 minutes at room temperature. The blot was developed using the ECLWestern Detection Kit (Cytiva) and quantified using iBright analysissoftware (Thermo Fisher Scientific). Images of Western blots are shownfor muscle tissues collected two-weeks following injections in FIGS. 22A(quadriceps), 23A (heart), and 24A (diaphragm), and quantification ofthe Western blot results are shown in FIGS. 22B (quadriceps), 23B(heart), and 24B (diaphragm). Images of western blots are shown formuscle tissues collected four-weeks following injections in FIGS. 22C(quadriceps), 23C (heart), and 25C (diaphragm), and quantification ofthe Western blot results are shown in FIGS. 22D (quadriceps), 23D(heart), and 24D (diaphragm). In each western blot, the standard curvewas generated using pooled protein from wild-type and mdx tissues, andthe percentage wild-type (% WT) protein in each standard indicates theamount of wild-type protein in the sample. FIGS. 22A-22D demonstratethat two- and four-weeks following administration, Ab-ASO facilitatedincreases in dystrophin protein in quadriceps to a greater extent thanunconjugated ASO. FIGS. 23A-23D demonstrate that two- and four-weeksfollowing administration, Ab-ASO facilitated increases in dystrophinprotein in heart muscle, whereas little to no wild-type dystrophin wasmeasured in heart muscle from mice treated with naked ASO. FIGS. 24A-24Ddemonstrate that two- and four-weeks following administration, Ab-ASOfacilitated increases in dystrophin protein in diaphragm muscle, whereaslittle to no wild-type dystrophin was measured in diaphragm muscle frommice treated with naked ASO.

Measurement of ASO content within tissues: Enzyme-linked immunosorbentassay (ELISA) was performed by coating NeutrAvidin coated plates with acapture probe specific to the ASO of interest. Proteinase K digestedtissue lysate was incubated on the coated plates to allow binding of theASO of interest to the capture probe. Plates were then washed andunbound capture probe was digested with micrococcal nuclease, followedby further washing and blocking. A Digoxigenin HRP-conjugated antibodywas added to bind to intact capture probe, then imaged using TMBsubstrate (R&D Systems, Inc.). Quantification was performed using astandard curve of known concentration diluted into skeletal musclematrix. The results demonstrate that administration of the Fab conjugateis able to achieve substantial accumulation of ASO within quadriceps(FIG. 25A), diaphragm (FIG. 25B), and heart (FIG. 25C), whereasadministration of naked ASO showed little or no ASO content in eachmuscle tissue. These results demonstrate that little or no ASO wasdetected in muscle tissues of mice administered saline or naked ASO,whereas administration of Ab-ASO resulted in measurable quantities ofASO in each of the tissues tested two- and four-weeks followingadministration.

Example 19. Conjugation of DMD Exon 53-Skipping Oligonucleotides toAnti-TfR1 Antibodies Improves their Potency

To test the effect of anti-TfR1 targeting on exon 53-skippingoligonucleotides, complexes were formed comprising an anti-TfR1 Fabantibody (3M12 VH4/Vk3) covalently linked to exon 53-skipping PMOs via alinker having the structure of formula (C). Two exon 53-skipping PMOswere used in this Example: exon 53 PMO-A, comprising the sequenceGTTGCCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 2256), and exon 53 PMO-B,comprising the sequence CCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 2257).

First, the exon 53-skipping PMOs alone were tested for their ability tofacilitate skipping of exon 53 following gymnotic uptake (i.e., withouttransfection agent or modification to confer muscle targeting). KM1328DMD patient cells which harbor a deletion of DMD exon 52 were treatedwith a range of concentrations of exon 53 PMO-A or exon 53 PMO-B, andexon 53 skipping was measured. As shown in FIG. 26 , exon 53 PMO-A wasabout 2-fold more potent than exon 53 PMO-B. Based on the dose responsecurves, it was calculated that a concentration of 2.5 μM of exon 53PMO-A or 4.7 μM of exon 53 PMO-B is required to achieve 50% skipping ofexon 53.

Next, complexes comprising the anti-TfR1 Fab covalently linked to eitherexon 53 PMO-A or exon 53 PMO-B (“anti-TfR1 Fab-ASO complex”) were testedfor their ability to facilitate skipping of exon 53 in KM1328 DMDpatient cells in comparison with the same PMOs not linked to an antibody(“naked ASO”). Cells were treated with the naked ASO at concentrationsof 0.16 μM, 0.32 μM, 0.63 μM, or 1.25 μM, or with the anti-TfR1 Fab-ASOcomplex at ASO equivalent concentrations of 0.16 μM, 0.32 μM, 0.63 μM,or 1.25 μM. As shown in FIG. 27 , the Fab-ASO complexes achieved greaterexon 53 skipping than did the naked ASO at each of the testedconcentrations, including achieving significantly improved exon 53skipping by exon 53 PMO-A at the lower doses tested (0.16 μM, 0.32 μM,and 0.63 μM). These results demonstrate that covalently linkingexon-skipping oligonucleotides to anti-TfR1 antibodies can facilitateexon-skipping activity at lower doses, thereby enabling efficacy of theoligonucleotides at lower doses.

Additional Embodiments

1. A complex comprising a muscle-targeting agent covalently linked to amolecular payload configured for promoting the expression or activity ofa DMD gene, wherein the muscle-targeting agent specifically binds to aninternalizing cell surface receptor on muscle cells, wherein the muscletargeting agent is a humanized antibody, wherein the antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 69; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 70;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 75;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 75;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 80.

2. The complex of embodiment 1, wherein the antibody comprises:

(i) a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(ii) a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(iii) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(iv) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VLcomprising the amino acid sequence of SEQ ID NO: 74;

(v) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(vi) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 74;

(vii) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(viii) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VLcomprising the amino acid sequence of SEQ ID NO: 78;

(ix) a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VLcomprising the amino acid sequence of SEQ ID NO: 80; or

(x) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VLcomprising the amino acid sequence of SEQ ID NO: 80.

3. The complex of embodiment 1 or embodiment 2, wherein the antibody isselected from the group consisting of a full-length IgG, a Fab fragment,a Fab′ fragment, a F(ab′)2 fragment, a scFv, and a Fv.

4. The complex of embodiment 3, wherein the antibody is a full-lengthIgG, optionally wherein the full-length IgG comprises a heavy chainconstant region of the isotype IgG1, IgG2, IgG3, or IgG4.

5. The complex of embodiment 4, wherein the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 84; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 86; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 87; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 88; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 88; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 91; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 91; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 92; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 94; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 92; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

6. The complex of embodiment 3, wherein the antibody is a Fab fragment.

7. The complex of embodiment 6, wherein the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 97; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 98; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 99; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 103; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

8. The complex of embodiment 6 or embodiment 7, wherein the antibodycomprises:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101;and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO:101; and a light chain comprising the amino acid sequence of SEQ ID NO:90;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO:102; and a light chain comprising the amino acid sequence of SEQ ID NO:93;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103;and a light chain comprising the amino acid sequence of SEQ ID NO: 95;or

(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102;and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

9. The complex of any one of embodiments 1 to 8, wherein the equilibriumdissociation constant (K_(D)) of binding of the antibody to thetransferrin receptor is in a range from 10⁻¹¹M to 10⁻⁶ M.

10. The complex of any one of embodiments 1 to 9, wherein the antibodydoes not specifically bind to the transferrin binding site of thetransferrin receptor and/or wherein the muscle-targeting antibody doesnot inhibit binding of transferrin to the transferrin receptor.

11. The complex of any one of embodiments 1 to 10, wherein the antibodyis cross-reactive with extracellular epitopes of two or more of a human,non-human primate and rodent transferrin receptor.

12. The complex of any one of embodiments 1 to 11, wherein the complexis configured to promote transferrin receptor mediated internalizationof the molecular payload into a muscle cell. 13. The complex of any oneof embodiments 1 to 12, wherein the antibody is a chimeric antibody,optionally wherein the chimeric antibody is a humanized monoclonalantibody.

14. The complex of any one of embodiments 1 to 13, wherein the antibodyis in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)₂ fragment,or Fv fragment.

15. The complex of any one of embodiments 1 to 14, wherein the molecularpayload is an oligonucleotide.

16. The complex of embodiment 15, wherein the oligonucleotide comprisesa sequence listed in Table 14.

16.1. The complex of embodiment 15, wherein the oligonucleotidecomprises any one of SEQ ID NO: 437-1241, or is complementary to any oneof SEQ ID NO: 1242-2046.

17. The complex of embodiment 16 or embodiment 16.1, wherein theoligonucleotide comprises a region of complementarity to a mutated DMDallele.

18. The complex of any one of embodiments 1 to 14, wherein the molecularpayload is a polypeptide.

19. The complex of embodiment 18, wherein the polypeptide is afunctional fragment of dystrophin protein.

20. The complex of any one of embodiments 15 to 17, wherein theoligonucleotide is configured to suppress a truncating mutation in a DMDallele by mono- or multi-exon skipping.

21. The complex of any one of embodiments 15 to 17, wherein theoligonucleotide promotes antisense-mediated exon skipping to producein-frame dystrophin mRNA.

22. The complex of embodiment 21, wherein the oligonucleotide promotesskipping of an exon of DMD in the range of exon 8 to exon 55, optionallyexon 23 to exon 53.

23. The complex of embodiment 22, wherein the oligonucleotide promotesskipping of exon 8, exon 23, exon 44, exon 45, exon 50, exon 51, exon52, exon 53, and/or exon 55.

24. The complex of embodiment 21, wherein the oligonucleotide promotesskipping of exon 51.

25. The complex of embodiment 24, wherein the oligonucleotide promotesskipping of multiple exons in the range of exon 44 to exon 53.

26. The complex of any one of embodiments 15 to 17 or 20 to 25, whereinthe oligonucleotide comprises at least one modified internucleotidelinkage.

27. The complex of embodiment 26, wherein the at least one modifiedinternucleotide linkage is a phosphorothioate linkage.

28. The complex of embodiment 27, wherein the oligonucleotide comprisesphosphorothioate linkages in the Rp stereochemical conformation and/orin the Sp stereochemical conformation.

29. The complex of embodiment 28, wherein the oligonucleotide comprisesphosphorothioate linkages that are all in the Rp stereochemicalconformation or that are all in the Sp stereochemical conformation.

30. The complex of any one of embodiments 15 to 17 or 20 to 29, whereinthe oligonucleotide comprises one or more modified nucleotides.

31. The complex of embodiment 30, wherein the one or more modifiednucleotides are 2′-modified nucleotides.

32. The complex of any one of embodiments 15 to 17 or 20 to 31, whereinthe oligonucleotide is a gapmer oligonucleotide that directs RNAseH-mediated cleavage of an miRNA that negatively regulates DMD expressionin a cell, optionally wherein the miRNA is miR-31.

33. The complex of embodiment 32, wherein the gapmer oligonucleotidecomprises a central portion of 5 to 15 deoxyribonucleotides flanked bywings of 2 to 8 modified nucleotides.

34. The complex of embodiment 33, wherein the modified nucleotides ofthe wings are 2′-modified nucleotides.

35. The complex of any one of embodiments 15 to 17 or 20 to 31, whereinthe oligonucleotide is a mixmer oligonucleotide.

36. The complex of embodiment 35, wherein the mixmer oligonucleotidepromotes exon skipping.

37. The complex of embodiment 35 or 36, wherein the mixmeroligonucleotide comprises two or more different 2′ modified nucleotides.

38. The complex of any one of embodiments 15 to 17 or 20 to 31, whereinthe oligonucleotide is an RNAi oligonucleotide that promotesRNAi-mediated cleavage of an miRNA that negatively regulates DMDexpression in a cell, optionally wherein the miRNA is miR-31.

39. The complex of embodiment 38, wherein the RNAi oligonucleotide is adouble-stranded oligonucleotide of 19 to 25 nucleotides in length.

40. The complex of embodiment 38 or 39, wherein the RNAi oligonucleotidecomprises at least one 2′ modified nucleotide.

41. The complex of any one of embodiments 31, 34, 37, or 40, whereineach 2′ modified nucleotide is selected from the group consisting of:2′-O-methyl, 2′-fluoro (2′-F), 2′-O-methoxyethyl (2′-MOE), and 2′,4′-bridged nucleotides.

42. The complex of embodiment 30, wherein the one or more modifiednucleotides are bridged nucleotides.

43. The complex of any one of embodiments 31, 34, 37, or 40, wherein atleast one 2′ modified nucleotide is a 2′,4′-bridged nucleotide selectedfrom: 2′,4′-constrained 2′-O-ethyl (cEt) and locked nucleic acid (LNA)nucleotides.

44. The complex of any one of embodiments 15 to 17 or 20 to 31, whereinthe oligonucleotide comprises a guide sequence for a genome editingnuclease.

45. The complex of any one of embodiments 15 to 17 or 20 to 31, whereinthe oligonucleotide is phosphorodiamidate morpholino oligomer.

46. The complex of any one of embodiments 1 to 45, wherein themuscle-targeting agent is covalently linked to the molecular payload viaa cleavable linker.

47. The complex of embodiment 46, wherein the cleavable linker isselected from: a protease-sensitive linker, pH-sensitive linker, andglutathione-sensitive linker.

48. The complex of embodiment 47, wherein the cleavable linker is aprotease-sensitive linker.

49. The complex of embodiment 48, wherein the protease-sensitive linkercomprises a sequence cleavable by a lysosomal protease and/or anendosomal protease.

50. The complex of embodiment 48, wherein the protease-sensitive linkercomprises a valine-citrulline dipeptide sequence.

51. The complex of embodiment 47, wherein the linker is pH-sensitivelinker that is cleaved at a pH in a range of 4 to 6.

52. The complex of any one of embodiments 1 to 45, wherein themuscle-targeting agent is covalently linked to the molecular payload viaa non-cleavable linker.

53. The complex of embodiment 52, wherein the non-cleavable linker is analkane linker.

54. The complex of any one of embodiments 1 to 53, wherein the antibodycomprises a non-natural amino acid to which the oligonucleotide iscovalently linked.

55. The complex of any one of embodiments 1 to 53, wherein the antibodyis covalently linked to the oligonucleotide via conjugation to a lysineresidue or a cysteine residue of the antibody.

56. The complex of embodiment 55, wherein the oligonucleotide isconjugated to the cysteine of the antibody via a maleimide-containinglinker, optionally wherein the maleimide-containing linker comprises amaleimidocaproyl or maleimidomethyl cyclohexane-1-carboxylate group.

57. The complex of any one of embodiments 1 to 56, wherein the antibodyis a glycosylated antibody that comprises at least one sugar moiety towhich the oligonucleotide is covalently linked.

58. The complex of embodiment 57, wherein the sugar moiety is a branchedmannose.

59. The complex of embodiment 57 or 58, wherein the antibody is aglycosylated antibody that comprises one to four sugar moieties each ofwhich is covalently linked to a separate oligonucleotide.

60. The complex of embodiment 57, wherein the antibody is afully-glycosylated antibody.

61. The complex of embodiment 57, wherein the antibody is apartially-glycosylated antibody.

62. The complex of embodiment 61, wherein the partially-glycosylatedantibody is produced via chemical or enzymatic means.

63. The complex of embodiment 61, wherein the partially-glycosylatedantibody is produced in a cell that is deficient for an enzyme in the N-or O-glycosylation pathway.

64. A method of delivering a molecular payload to a cell expressingtransferrin receptor, the method comprising contacting the cell with thecomplex of any one of embodiments 1 to 63.

65. A method of promoting the expression or activity of a DMD protein ina cell, the method comprising contacting the cell with the complex ofany one of embodiments 1 to 63 in an amount effective for promotinginternalization of the molecular payload to the cell.

66. The method of embodiment 65, wherein the cell is in vitro.

67. The method of embodiment 65, wherein the cell is in a subject.

68. The method of embodiment 67, wherein the subject is a human.

69. A method of treating a subject having a mutated DMD allele that isassociated with a dystrophinopathy, the method comprising administeringto the subject an effective amount of the complex of any one ofembodiments 1 to 63.

70. A method of promoting skipping of an exon of a DMD mRNA transcriptin a cell, the method comprising administering to the cell an effectiveamount of the complex of any one of embodiments 1 to 63.

71. The method of embodiment 70, wherein the method promotes skipping ofexon 8, exon 23, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53,and/or exon 55 of the DMD mRNA transcript.

72. The method of embodiment 70 or 71, wherein the method promotesskipping of exon 51 of the DMD mRNA transcript.

73. A complex comprising an anti-transferrin receptor (TfR) antibodycovalently linked to a molecular payload configured for promoting theexpression or activity of a DMD gene, wherein the antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 76; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 75;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 69; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 74;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 75;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 80.

73. A complex comprising an anti-transferrin receptor (TfR) antibodycovalently linked to a molecular payload configured for promoting theexpression or activity of a DMD gene, wherein the anti-TfR antibody hasundergone pyroglutamate formation resulting from a post-translationalmodification.

EQUIVALENTS AND TERMINOLOGY

The disclosure illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure. Thus, it should be understood that although thepresent disclosure has been specifically disclosed by preferredembodiments, optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups or other grouping of alternatives, thoseskilled in the art will recognize that the disclosure is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group or other group.

It should be appreciated that, in some embodiments, sequences presentedin the sequence listing may be referred to in describing the structureof an oligonucleotide or other nucleic acid. In such embodiments, theactual oligonucleotide or other nucleic acid may have one or morealternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide ora DNA counterpart of an RNA nucleotide) and/or (e.g., and) one or moremodified nucleotides and/or (e.g., and) one or more modifiedinternucleotide linkages and/or (e.g., and) one or more othermodification compared with the specified sequence while retainingessentially same or similar complementary properties as the specifiedsequence.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description.

The inventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-28. (canceled)
 29. A composition comprising complexes comprising ananti-transferrin receptor antibody covalently linked to at least oneoligonucleotide, wherein the antibody is a Fab and comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 101 and a lightchain comprising the amino acid sequence of SEQ ID NO: 90, and whereineach anti-TfR antibody of the complexes is on average covalently linkedto 1 to 3 oligonucleotides, and wherein the oligonucleotide inducesdystrophin exon 51 skipping.
 30. The composition of claim 29, whereinthe heavy chain of the antibody comprises an N-terminal pyroglutamate.31. The composition of claim 29, wherein the equilibrium dissociationconstant (K_(D)) of binding of the antibody to the transferrin receptoris in a range from 10⁻¹¹M to 10⁻⁶ M.
 32. The composition of claim 29,wherein the oligonucleotide is 15-35 nucleotides in length.
 33. Thecomposition of claim 29, wherein the oligonucleotide comprises a regionof complementarity to a dystrophin RNA, wherein the region ofcomplementarity is 12-35 nucleotides in length.
 34. The composition ofclaim 29, wherein the oligonucleotide comprises a region ofcomplementarity to the target sequence of an oligonucleotide as setforth in any one of SEQ ID NOs: 333-357, 745, and 2254, wherein theregion of complementarity is 12-35 nucleotides in length.
 35. Thecomposition of claim 29, wherein the oligonucleotide comprises thenucleotide sequence of any one of SEQ ID NOs: 333-357, 745, and 2254,wherein any one or more of the thymine bases (T's) in theoligonucleotide may optionally be a uracil base (U), and wherein any oneor more of the uracil bases (U's) in the oligonucleotide may optionallybe a thymine base (T).
 36. The composition of claim 29, wherein theoligonucleotide comprises one or more modified nucleosides.
 37. Thecomposition of claim 29, wherein the oligonucleotide is aphosphorodiamidate morpholino oligomer.
 38. The composition of claim 29,wherein the antibody and the molecular payload are covalently linked viaa linker.
 39. The composition of claim 38, wherein the linker comprisesa cleavable linker.
 40. The composition of claim 39, wherein the linkercomprises a valine-citrulline sequence.
 41. The composition of claim 29,wherein the complex comprises a structure of:

wherein n is 3 and m is 4, wherein L1 comprises a spacer that is asubstituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted carbocyclylene,substituted or unsubstituted heterocyclylene, substituted orunsubstituted arylene, substituted or unsubstituted heteroarylene, —O—,—N(R^(A))—, —S—, —C(═O)—, —C(═O)O—, —C(═O)NR^(A)—, —NR^(A)C(═O)—,—NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—, —NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—,—OC(═O)—, —OC(═O)O—, —OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, ora combination thereof, wherein each R^(A) is independently hydrogen orsubstituted or unsubstituted alkyl.
 42. The composition of claim 41,wherein L1 comprises a structure of:

wherein the piperazine moiety links to the oligonucleotide, wherein L2comprises


43. The composition of claim 42, wherein L2 comprises


44. A method of inducing dystrophin exon 51 skipping in muscle cells ofa subject, the method comprising administering to the subject thecomposition of claim
 29. 45. The method of claim 44, wherein the subjectis human.
 46. The method of claim 44, wherein the subject is acynomolgus.
 47. The method of claim 44, wherein the subject expresses adystrophin pre-mRNA comprising a frameshift mutation.
 48. The method ofclaim 44, wherein the complex is intravenously administered to thesubject.
 49. The method of claim 44, wherein the subject is amenable todystrophin exon 51 skipping.
 50. The method of claim 44, wherein thesubject has Duchenne muscular dystrophy.
 51. The method of claim 44,wherein the heavy chain of the antibody comprises an N-terminalpyroglutamate.